Lysophosphatidic acid (LPA) is both a potential marker and a therapeutic target for ovarian cancer. It is critical to identify the sources of elevated LPA levels in ascites and blood of patients with ovarian cancer. We show here that human peritoneal mesothelial cells constitutively produce LPA, which accounts for a significant portion of the chemotactic activity of the conditioned medium from peritoneal mesothelial cells to ovarian cancer cells. Both production of LPA by peritoneal mesothelial cells and the chemotactic activity in the conditioned medium can be blocked by HELSS [an inhibitor of the calcium-independent phospholipase A 2 (iPLA 2 )] and AACOCF 3 [an inhibitor of both cytosolic PLA 2 (cPLA 2 ) and iPLA 2 ]. Moreover, cell-based enzymatic activity assays for PLA 2 indicate that peritoneal mesothelial cells have strong constitutive PLA 2 activity. Receptors for LPA, LPA 2 , and LPA 3 are involved in the conditioned medium-induced chemotactic activity. Invasion of ovarian cancer cells into peritoneal mesothelial cells has also been analyzed and shown to require PLA 2 , LPA receptors, and the mitogen-activated protein/ extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase signaling pathway. Thus, we show here, for the first time, that human peritoneal mesothelial cells constitutively produce bioactive lipid signaling molecules, such as LPA, via iPLA 2 and/or cPLA 2 activities. Conditioned medium from peritoneal mesothelial cells stimulate migration, adhesion, and invasion of ovarian cancer cells, and may play similar roles in vivo.

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Calcium-independent phospholipase A 2 (iPLA 2 ) plays a pivotal role in phospholipid remodeling and many other biological processes, including inflammation and cancer development. iPLA 2 can be activated by caspase-3 via a proteolytic process in apoptotic cells. In this study we identify novel signaling and functional loops of iPLA 2 activation leading to migration of non-apoptotic human ovarian cancer cells. The extracellular matrix protein, laminin-10/11, but not collagen I, induces integrin-and caspase-3-dependent cleavage and activation of overexpressed and endogenous iPLA 2 . The truncated iPLA 2 (amino acids 514 -806) generates lysophosphatidic acid and arachidonic acid. Arachidonic acid is important for enhancing cell migration toward laminin-10/11. Lysophosphatidic acid activates Akt that in turn acts in a feedback loop to block the cleavage of poly-(ADP-ribose) polymerase and DNA fragmentation factor as well as prevent apoptosis. By using pharmacological inhibitors, blocking antibodies, and genetic approaches (such as point mutations, dominant negative forms of genes, and siRNAs against specific targets), we show that ␤ 1 , but not ␤ 4 , integrin is involved in iPLA 2 activation and cell migration to laminin-10/ 11. The role of caspase-3 in iPLA 2 activation and cell migration are supported by several lines of evidence. 1) Point mutation of Asp 513 (a cleavage site of caspase-3 in iPLA 2 ) to Ala blocks laminin-10/11-induced cleavage and activation of overexpressed iPLA 2 , whereas mutation of Asp 733 to Ala has no such effect, 2) treatment of inhibitors or a small interfering RNA against caspase-3 results in decreased cell migration toward laminin-10/11, and 3) selective caspase-3 inhibitor blocks cleavage of endogenous iPLA 2 induced by laminin-10/11. Importantly, small interfering RNA-mediated down-regulation of endogenous iPLA 2 expression in ovarian carcinoma HEY cells results in decreased migration toward laminin, suggesting that our findings are pathophysiologically important.

Objective To develop claims‐based measures of comprehensiveness of primary care physicians (PCPs) and summarize their associations with health care utilization and cost. Data Sources and Study Setting A total of 5359 PCPs caring for over 1 million Medicare fee‐for‐service beneficiaries from 1404 practices. Study Design We developed Medicare claims‐based measures of physician comprehensiveness (involvement in patient conditions and new problem management) and used a previously developed range of services measure. We analyzed the association of PCPs’ comprehensiveness in 2013 with their beneficiaries’ emergency department, hospitalizations rates, and ambulatory care‐sensitive condition (ACSC) admissions (each per 1000 beneficiaries per year), and Medicare expenditures (per beneficiary per month) in 2014, adjusting for beneficiary, physician, practice, and market characteristics, and clustering. Principal Findings Each measure varied across PCPs and had low correlation with the other measures—as intended, they capture different aspects of comprehensiveness. For patients whose PCPs’ comprehensiveness score was at the 75th vs 25th percentile (more vs less comprehensive), patients had lower service use (P < 0.05) in one or more measures: involvement with patient conditions: total Medicare expenditures, −$17.4 (−2.2 percent); hospitalizations, −5.5 (−1.9 percent); emergency department (ED) visits, −16.3 (−2.4 percent); new problem management: total Medicare expenditures, −$13.3 (−1.7 percent); hospitalizations, −7.0 (−2.4 percent); ED visits, −19.7 (−2.9 percent); range of services: ED visits, −17.1 (−2.5 percent). There were no significant associations between the comprehensiveness measures and ACSC admission rates. Conclusions These measures demonstrate strong content and predictive validity and reliability. Medicare beneficiaries of PCPs providing more comprehensive care had lower hospitalization rates, ED visits, and total Medicare expenditures.

Objectives. To develop and evaluate a board game designed to increase students' enjoyment of learning metabolic pathways; their familiarity with pathway reactions, intermediates, and regulation; and, their understanding of how pathways relate to one another and to selected biological conditions. Design. The board game, entitled Race to Glucose, was created as a team activity for first-year pharmacy students in the biochemistry curriculum. Assessment. A majority of respondents agreed that the game was helpful for learning regulation, intermediates, and interpathway relationships but not for learning reactions, formation of energetic molecules, or relationships, to biological conditions. There was a significant increase in students' scores on game-related examination questions (68.8% pretest vs. 81.3% posttest), but the improvement was no greater than that for examination questions not related to the game (12.5% vs. 10.9%). Conclusion. First-year pharmacy students considered Race to Glucose to be an enjoyable and helpful tool for learning intermediates, regulation, and interpathway relationships.

PLA (phospholipases A) are important mediators of cell signaling, generating bioactive fatty acids and LPLs (lysophospholipids). PLA products having different head groups can initiate vastly different types of signaling. Fluorogenic analogues of the PLs (phospholipids) PA (phosphatidic acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), and PG (phosphatidylglycerol) were synthesized as PLA substrates for rapidly determining in real time the influence of head group modifications on cell signaling both in vitro and in cells. Enzyme-assisted remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom PLA 2 and transphosphatidylation with a particular PLD (phospholipase D) were central steps in the preparation of these enzymatic probes. The resulting fluorogenic Dabcyl- and BODIPY-containing PL analogues, DBPA, DBPC, DBPE, and DBPG, were used in mixed micelle assays to determine PLA 2 kinetics. Next, the assays were used to determine the X i (50) value of a common PLA 2 inhibitor. Finally, the head group selectivities of a series of commercially available PLA 2 enzymes were readily established using the DBPLs (Dabcyl-BODIPY PLs) as substrates.

Fluorogenic analogues of phosphatidylcholine and lysophosphatidylcholine, DDPB and lysoDDPB, were synthesized by an enzyme-assisted strategy. The analogues were evaluated as substrates for phospholipases C and D, and lysophospholipase D. DDPB was cleaved by bacterial and plant phospholipase D (PLD) enzymes and represents the first direct fluorogenic substrate for real-time measurement of PLD activity. Both fluorogenic substrates have potential in screening for PLD and PC-PLC inhibitors, and for monitoring spatiotemporal changes in PLD activity in cells.PLC, PLD, and lysoPLD are three phospholipases that catalyze hydrolysis of phospholipid (PL) head groups. PLC catalyzes the hydrolysis of the PL phosphodiester glyceryl P-O bond to give diacylglycerol and a phosphomonoester. PLD cleaves the head group P-O bond of the phosphodiester linkage, to produce phosphatidic acid (PA) and an alcohol. LysoPLD catalyzes the same reaction as PLD, but is selective for lysophospholipids. Most PLDs also catalyze transphosphatidylation, 1 in which a primary alcohol replaces water as the cleaving nucleophile.PLC isozymes selectively hydrolyze PLs with either inositol or choline headgroups. There are at least eleven different phosphoinositide-selective PLCs (PI-PLC), 2 two putative phosphatidylcholine-selective PLCs (PC-PLCs) 3 in mammals, and one PC-PLC in plants. 4 PLD isozymes found in mammals are involved in a diversity of normal and disease-related biological processes. 5-7 The PLD enzymes found in mammals, plants, and some bacteria are called "HKD PLD" because they share a consensus amino acid sequence (HxKx 4 Dx 6 GG/S), and employ a common catalytic mechanism involving a covalent histidine intermediate. 8 The "non-HKD PLD" enzymes lack an HKD consensus sequence, and have a catalytic mechanism in which metal ions are required to position the PL substrate and activate the attacking nucleophile. 9 S. chromofuscus PLD (scPLD) is an archetypical non-HKD PLD. Similar to the Email: Glenn.Prestwich@hsc.utah.edu. We report here the synthesis of fluorogenic PC and lysoPC analogues that contain a fluorescence quencher (dabcyl, a.k.a p-methyl red) at each acyl chain terminus, and a fluorophore appended to the PL head group through a choline-mimetic linker. These PL analogues, denoted DDPB and lysoDDPB, were evaluated in microtiter plate assays as substrates for lysoPLD, scPLD, PLC, and phospholipase A 2 (PLA 2 ), as well as several commercially-available HKD PLD enzymes. NIH Public AccessDDPB and lysoDDPB were synthesized efficiently as illustrated in Schemes 1 and 2. The lysophosphatidylcholine intermediate 2 containing the shorter dabcyl acyl chain was obtained a selective monoacylation of the commercially-available glycerol phosphocholine, 1, followed by installation of the longer-chain dabcyl quencher at the sn-2 position (4, Scheme 1). While the longer, dodecanoyl acyl chain at sn-2 improved the lipophilicity of the analogue over compounds with two hexanoyl chains, adding a second C 12 linker at sn-1 afforded a less soluble ...

Metformin (MET) is increasingly implicated in reducing the incidence of multiple cancer types in patients with diabetes. However, similar effects of MET in non-diabetic women with endometrial cancer (EC) remain unknown. In a pilot study, obese non-diabetic women diagnosed with type 1, grade 1/2 EC, and consenting to participate were randomly assigned to receive MET or no MET (control (CON)) during the pre-surgical window between diagnosis and hysterectomy. Endometrial tumors obtained at surgery (MET, n = 4; CON, n = 4) were analyzed for proliferation (Ki67), apoptosis (TUNEL), and nuclear expression of ERα, PGR, PTEN, and KLF9 proteins in tumor glandular epithelial (GE) and stromal (ST) cells. The percentages of immunopositive cells for PGR and for KLF9 in GE and for PTEN in ST were higher while those for ERα in GE but not ST were lower, in tumors of MET vs. CON patients. The numbers of Ki67-and TUNEL-positive cells in tumor GE and ST did not differ between groups. In human Ishikawa endometrial cancer cells, MET treatment (60 μM) decreased cell numbers and elicited distinct temporal changes in ESR1, KLF9, PGR, PGR-B, KLF4, DKK1, and other tumor biomarker mRNA levels. In the context of reduced KLF9 expression (by siRNA targeting), MET rapidly amplified PGR, PGR-B, and KLF4 transcript levels. Our findings suggest that MET acts directly in EC cells to modify steroid receptor expression and signaling network and may constitute a preventative strategy against EC in high-risk non-diabetic women.