Ramelteon for Prevention of Postoperative Delirium: A Randomized Controlled Trial in Patients Undergoing Elective Pulmonary Thromboendarterectomy.

OBJECTIVES: To assess the efficacy of ramelteon in preventing delirium, an acute neuropsychiatric condition associated with increased morbidity and mortality, in the perioperative, ICU setting. DESIGN: Parallel-arm, randomized, double-blinded, placebo-controlled trial. SETTING: Academic medical center in La Jolla, California. PATIENTS: Patients greater than or equal to 18 years undergoing elective pulmonary thromboendarterectomy. INTERVENTIONS: Ramelteon 8 mg or matching placebo starting the night prior to surgery and for a maximum of six nights while in the ICU. MEASUREMENTS AND MAIN RESULTS: Incident delirium was measured twice daily using the Confusion Assessment Method-ICU. The safety outcome was coma-free days assessed by the Richmond Agitation-Sedation Scale. One-hundred twenty participants were enrolled and analysis completed in 117. Delirium occurred in 22 of 58 patients allocated to placebo versus 19 of 59 allocated to ramelteon (relative risk, 0.8; 95% CI, 0.5-1.4; p = 0.516). Delirium duration, as assessed by the number of delirium-free days was also similar in both groups (placebo median 2 d [interquartile range, 2-3 d] vs ramelteon 3 d [2-5 d]; p = 0.181). Coma-free days was also similar between groups (placebo median 2 d [interquartile range, 1-3 d] vs ramelteon 3 d [2-4 d]; p = 0.210). We found no difference in ICU length of stay (median 4 d [interquartile range, 3-5 d] vs 4 d [3-6 d]; p = 0.349), or in-hospital mortality (four vs three deaths; relative risk ratio, 0.7; 95% CI, 0.2-3.2; p = 0.717), all placebo versus ramelteon, respectively. CONCLUSIONS: Ramelteon 8 mg did not prevent postoperative delirium in patients admitted for elective cardiac surgery.

Amphiphilic suramin dissolves Matrigel, causing an 'inhibition' artefact within in vitro angiogenesis assays.

The field of study concerning promotion and/or inhibition of angiogenesis has gathered much attention in the scientific community. A great deal of work has been invested towards defining reproducible assays to gauge for promotion or inhibition of angiogenesis in response to drug treatments or growth conditions. Two common components of these assays were noted by our group to have an unexpected and previously unreported interaction. Suramin is a commercially available compound, commonly used as a positive control for in vitro angiogenic inhibition assays. Matrigel is a popular extracellular substrate that supports angiogenic network formation when endothelial cells are cultured on its surface. However, our group demonstrated that suramin alone (without the presence of cells) will actively dissolve Matrigel, causing the extracellular matrix to transition from the gel-like physical state to a more liquid state. This causes cells on the Matrigel to congregate and sink to the bottom of the well. Therefore, previous observations of inhibition of endothelial cell angiogenesis through the incubation with suramin (including previous observations made by our group) are, largely, an artefact caused by suramin and matrix interaction rather than suramin and cells interaction, as previously reported. Our results suggest that the presence of sulphate groups and amphiphilic properties of suramin are likely responsible for the disruption of the matrix layer. We believe that this information is of prime importance to anyone using similar in vitro models, or employing suramin in any therapy or drug development assays.

Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening.

BACKGROUND: Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting 'normalization' of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell-cell and cell-matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation. HUVECs (human umbilical-vein endothelial cells) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by six to eight passages in culture, making stable integration of fluorescent markers and large-scale expansion for HTS problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized HMECs (human microvascular endothelial cell). We then evaluated HMEC cultures, both alone and co-cultured with an EMC (epicardial mesothelial cell) line that contributes vascular smooth muscle cells, to determine the suitability for HTS or HCS. RESULTS: The endothelial and epicardial lines were engineered to express a panel of nuclear- and cytoplasm-localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 h after plating on Matrigel, the cells migrated and coalesced into networks of vessel-like structures. If co-cultured with EMCs, the branches formed cylindrical-shaped structures of HMECs surrounded by EMC derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds. CONCLUSIONS: HMEC-based lines retain most of the angiogenic features of primary endothelial cells and yet possess long-term stability and ease of culture, making them intriguing candidates for large-scale primary HCS and HTS (of ~10000-1000000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial-derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.