Subcellular imaging of lipids and sugars using genetically encoded proximity sensors.

Live cell imaging of lipids and other metabolites is a long-standing challenge in cell biology. Bioorthogonal labeling tools allow for the conjugation of fluorophores to several phospholipid classes, but cannot discern their trafficking between adjacent organelles or asymmetry across individual membrane leaflets. Here we present fluorogen-activating coincidence sensing (FACES), a chemogenetic tool capable of quantitatively imaging subcellular lipid pools and reporting their transbilayer orientation in living cells. FACES combines bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs) for reversible proximity sensing of conjugated molecules. We first validate this approach for quantifying discrete phosphatidylcholine pools in the ER and mitochondria that are trafficked by lipid transfer proteins. We then show that transmembrane domain-containing FAPs can be used to reveal the membrane asymmetry of multiple lipid classes that are generated in the trans-Golgi network. Lastly, we demonstrate that FACES is a generalizable tool for subcellular bioorthogonal imaging by measuring changes in mitochondrial N -acetylhexosamine levels. These results demonstrate the use of fluorogenic tags for spatially-defined molecular imaging.

Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology.

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.

Reprogramming sphingolipid glycosylation is required for endosymbiont persistence in Medicago truncatula.

Plant endosymbiosis relies on the development of specialized membranes that encapsulate the endosymbiont and facilitate nutrient exchange. However, the identity and function of lipids within these membrane interfaces is largely unknown. Here, we identify GLUCOSAMINE INOSITOL PHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) as a sphingolipid glycosyltransferase highly expressed in Medicago truncatula root nodules and roots colonized by arbuscular mycorrhizal (AM) fungi and further demonstrate that this enzyme functions in the synthesis of N-acetyl-glucosamine-decorated glycosyl inositol phosphoryl ceramides (GIPCs) in planta. MtGINT1 expression was developmentally regulated in symbiotic tissues associated with the development of symbiosome and periarbuscular membranes. RNAi silencing of MtGINT1 did not affect overall root growth but strongly impaired nodulation and AM symbiosis, resulting in the senescence of symbiosomes and arbuscules. Our results indicate that, although M. truncatula root sphingolipidome predominantly consists of hexose-decorated GIPCs, local reprogramming of GIPC glycosylation by MtGINT1 is required for the persistence of endosymbionts within the plant cell.

Draft Genome Sequence of Agrobacterium fabrum ARqua1.

Agrobacterium fabrum ARqua1 is a hybrid of Agrobacterium fabrum C58C bearing the megaplasmid pRiA4b. ARqua1 is used by many plant researchers to generate transgenic roots. The draft genome of ARqua1 includes a 249,350-bp contig that likely covers all of pRiA4b, and it will be a valuable resource to plant biologists.

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism.

Despite intensive study, plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remains unvalidated. Recently we described a missing step in the D-lysine catabolism of Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (2HG) via hydroxyglutarate synthase (HglS), a DUF1338 family protein. Here we solve the structure of HglS to 1.1 Å resolution in substrate-free form and in complex with 2OA. We propose a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O2-dependent manner. Specificity is mediated by a single arginine, highly conserved across most DUF1338 proteins. An Arabidopsis thaliana HglS homolog coexpresses with known lysine catabolism enzymes, and mutants show phenotypes consistent with disrupted lysine catabolism. Structural and biochemical analysis of Oryza sativa homolog FLO7 reveals identical activity to HglS despite low sequence identity. Our results suggest DUF1338-containing enzymes catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes.

Agrobacterium tumefaciens: A Bacterium Primed for Synthetic Biology.

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.

One-year results of the ANCHOR trial of EndoAnchors for the prevention and treatment of aortic neck complications after endovascular aneurysm repair.

OBJECTIVES: EndoAnchors have been used to address proximal aortic neck complications including type Ia endoleaks and endograft migration after endovascular aortic aneurysm repair (EVAR). METHODS: The study population included 100 patients with one-year follow-up in the ANCHOR study. A primary cohort (N = 73) comprised patients who underwent EndoAnchor implantation at the time of an initial EVAR and a Revision cohort (N = 27) included patients treated remote from EVAR. A hostile neck was defined for neck length <10 mm, neck diameter >28 mm, angulation >60°, conical configuration or significant mural thrombus or calcium. RESULTS: Baseline anatomy included neck length of 17 ± 14 mm, diameter of 27 ± 5 mm, and angulation of 35 ± 18°; 83% of patients had hostile necks. Over 18 ± 4 months of clinical follow-up, six patients (6%) underwent aneurysm-related reinterventions. There were no aneurysm ruptures. Over 13 ± 2 months of imaging follow-up, freedom from type Ia endoleak was 95% in the Primary Arm and 77% in the Revision Arm (P = .006). Aneurysm sacs regressed > 5 mm within one year in 45% of the Primary cases and in 25% of the Revisions. Aneurysm expansion > 5 mm occurred in one revision patient. CONCLUSION: Despite a high frequency of hostile neck anatomy, proximal neck complications were relatively infrequent after EndoAnchor use.

Midterm outcome of EndoAnchors for the prevention of endoleak and stent-graft migration in patients with challenging proximal aortic neck anatomy.

PURPOSE: To explore the use of EndoAnchors as an adjunct to endovascular abdominal aortic aneurysm repair for prevention of proximal neck complications in patients with challenging neck anatomy. METHODS: Over a 28-month period, 208 patients (159 men; mean age 72±8 years) were enrolled in the ANCHOR prospective, multicenter registry (ClinicalTrials.gov; identifier NCT01534819) for prophylaxis against proximal neck complications. Patients were eligible when, in the opinion of the investigators, they were at increased risk for type Ia endoleak or migration owing to a hostile neck (length <10 mm, diameter >28 mm, angulation >60°, mural thrombus or calcium >2 mm in thickness or >180° in circumference, or conical shape). Overall, 123/157 (78.3%) patients met the criteria for a hostile neck according to core laboratory assessment of 157 adequate preoperative computed tomographic (CT) images. RESULTS: Implantation of EndoAnchors was technically successful in 204/208 (98.1%) patients. The frequency of fracture was 0.3% (3/1118); there were no clinical sequelae associated with the fractures. Over the mean 14-month follow-up, 95.2% of patients were alive, and no deaths were attributable to EndoAnchors. There were no ruptures, migrations, or open surgical conversions. Aneurysm-related reinterventions were performed in 8 (3.8%) patients. Among 130 patients with postprocedure contrast CT studies, core laboratory analysis identified 2 (1.5%) patients with type Ia endoleaks. Aneurysm sac diameter decreased >5 mm in 42.9% of patients with CT scans at or beyond 1 year; 1.6% of patients developed sac enlargement >5 mm. CONCLUSION: Prophylactic EndoAnchor use for challenging aortic neck anatomy was associated with satisfactory midterm results.

Outcome-based anatomic criteria for defining the hostile aortic neck.

OBJECTIVE: There is abundant evidence linking hostile proximal aortic neck anatomy to poor outcome after endovascular aortic aneurysm repair (EVAR), yet the definition of hostile anatomy varies from study to study. This current analysis was undertaken to identify anatomic criteria that are most predictive of success or failure at the aortic neck after EVAR. METHODS: The study group comprised 221 patients in the Aneurysm Treatment using the Heli-FX Aortic Securement System Global Registry (ANCHOR) clinical trial, a population enriched with patients with challenging aortic neck anatomy and failure of sealing. Imaging protocols were not protocol specified but were performed according to the institution's standard of care. Core laboratory analysis assessed the three-dimensional centerline-reformatted computed tomography scans. Failure at the aortic neck was defined by type Ia endoleak occurring at the time of the initial endograft implantation or during follow-up. Receiver operating characteristic curve analysis was used to assess the value of each anatomic measure in the classification of aortic neck success and failure and to identify optimal thresholds of discrimination. Binary logistic regression was performed after excluding highly intercorrelated variables, creating a final model with significant predictors of outcome after EVAR. RESULTS: Among the 221 patients, 121 (54.8%) remained free of type Ia endoleak and 100 (45.2%) did not. Type Ia endoleaks presented immediately after endograft deployment in 58 (58.0%) or during follow-up in 42 (42.0%). Receiver operating characteristic curve analysis identified 12 variables where the classification of patients with type Ia endoleak was significantly more accurate than chance alone. Increased aortic neck diameter at the lowest renal artery (P = .013) and at 5 mm (P = .008), 10 mm (P = .008), and 15 mm (P = .010) distally; aneurysm sac diameter (P = .001), common iliac artery diameters (right, P = .012; left, P = .032), and a conical (P = .049) neck configuration were predictive of endoleak. By contrast, increased aortic neck length (P = .050), a funnel-shaped aortic neck (P = .036), and neck mural thrombus content, as measured by average thickness (P = .044) or degrees of circumferential coverage (P = .029), were protective against endoleak. Binary logistic regression identified three variables independently predictive of type Ia endoleak. Neck diameter at the lowest renal artery (P = .002, cutpoint 26 mm) and neck length (P = .017, cutpoint 17 mm) were associated with endoleak, whereas some mural neck thrombus content was protective (P = .001, cutpoint 11° of circumferential coverage). CONCLUSIONS: A limited number of independent anatomic variables are predictive of type Ia endoleak after EVAR, including aortic neck diameter and aortic neck length, whereas mural thrombus in the neck is protective. This study suggests that anatomic measures with identifiable threshold cutpoints should be considered when defining the hostile aortic neck and assessing the risk of complications after EVAR.

Telemedicine-based collaborative care for posttraumatic stress disorder: a randomized clinical trial.

IMPORTANCE: Posttraumatic stress disorder (PTSD) is prevalent, persistent, and disabling. Although psychotherapy and pharmacotherapy have proven efficacious in randomized clinical trials, geographic barriers impede rural veterans from engaging in these evidence-based treatments. OBJECTIVE: To test a telemedicine-based collaborative care model designed to improve engagement in evidence-based treatment of PTSD. DESIGN, SETTING, AND PARTICIPANTS: The Telemedicine Outreach for PTSD (TOP) study used a pragmatic randomized effectiveness trial design with intention-to-treat analyses. Outpatients were recruited from 11 Department of Veterans Affairs (VA) community-based outpatient clinics serving predominantly rural veterans. Inclusion required meeting diagnostic criteria for current PTSD according to the Clinician-Administered PTSD Scale. Exclusion criteria included receiving PTSD treatment at a VA medical center or a current diagnosis of schizophrenia, bipolar disorder, or substance dependence. Two hundred sixty-five veterans were enrolled from November 23, 2009, through September 28, 2011, randomized to usual care (UC) or the TOP intervention, and followed up for 12 months. INTERVENTIONS: Off-site PTSD care teams located at VA medical centers supported on-site community-based outpatient clinic providers. Off-site PTSD care teams included telephone nurse care managers, telephone pharmacists, telepsychologists, and telepsychiatrists. Nurses conducted care management activities. Pharmacists reviewed medication histories. Psychologists delivered cognitive processing therapy via interactive video. Psychiatrists supervised the team and conducted interactive video psychiatric consultations. MAIN OUTCOMES AND MEASURES: The primary outcome was PTSD severity as measured by the Posttraumatic Diagnostic Scale. Process-of-care outcomes included medication prescribing and regimen adherence and initiation of and adherence to cognitive processing therapy. RESULTS: During the 12-month follow-up period, 73 of the 133 patients randomized to TOP (54.9%) received cognitive processing therapy compared with 16 of 132 randomized to UC (12.1%) (odds ratio, 18.08 [95% CI, 7.96-41.06]; P < .001). Patients in the TOP arm had significantly larger decreases in Posttraumatic Diagnostic Scale scores (from 35.0 to 29.1) compared with those in the UC arm (from 33.5 to 32.1) at 6 months (ß = -3.81; P = .002). Patients in the TOP arm also had significantly larger decreases in Posttraumatic Diagnostic Scale scores (from 35.0 to 30.1) compared with those in the UC arm (from 33.5 to 31.7) at 12 months (ß = -2.49; P=.04). There were no significant group differences in the number of PTSD medications prescribed and adherence to medication regimens were not significant. Attendance at 8 or more sessions of cognitive processing therapy significantly predicted improvement in Posttraumatic Diagnostic Scale scores (ß = -3.86 [95% CI, -7.19 to -0.54]; P = .02) and fully mediated the intervention effect at 12 months. CONCLUSIONS AND RELEVANCE: Telemedicine-based collaborative care can successfully engage rural veterans in evidence-based psychotherapy to improve PTSD outcomes. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00821678.

A 28-day oral toxicity evaluation of small interfering RNAs and a long double-stranded RNA targeting vacuolar ATPase in mice.

New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48 mg/kg/day) and the 218 bp dsRNA (64 mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

Analysis of EndoAnchors for endovascular aneurysm repair by indications for use.

OBJECTIVE: The proximal aortic neck remains one of the challenges of endovascular aneurysm repair (EVAR), and the risk of type Ia endoleak and endograft migration is increased in patients with short, large-diameter, or highly angulated necks. EndoAnchors have been used as an adjunct to EVAR in such patients, and the aim of this study was to assess their benefit analyzed by indication for use. METHODS: During a 2-year period, 319 patients were enrolled at 43 sites in the Aneurysm Treatment Using the Heli-FX Aortic Securement System Global Registry (ANCHOR) study. This prospective, multinational, real-world analysis of EndoAnchors comprised two groups of patients, those undergoing first-time EVAR (primary arm, 242) and those with proximal neck complications remote from the time of an initial endograft implantation (revision arm, 77). The primary arm was further subdivided into patients undergoing prophylactic EndoAnchor use for hostile proximal neck anatomy (178), with a type Ia endoleak evident during initial endograft deployment (60), and in conjunction with extender cuffs after unsatisfactory endograft deployment distally in the neck (four). The revision arm was subdivided into patients presenting with a type Ia endoleak alone (45), endograft migration alone (11), and migration with endoleak (21). Technical success was site reported as satisfactory deployment of the desired number of EndoAnchors without fracture or loss of integrity. Procedural success was defined as technical success without type Ia endoleak at completion arteriography. Core laboratory analysis was performed on 249 baseline and 192 follow-up computed tomographic studies, 66 of which were available within the 1-year window. RESULTS: Technical and procedural success rates were highest in the prophylactically treated subset (172 of 178; 96.6%). Whereas the technical success of EndoAnchor deployment was also high in the other subsets, residual type Ia endoleaks were more frequent at completion angiography when the indication for EndoAnchor use was type Ia endoleak, both in the primary arm (17 of 60; 28%) and in the revision arm (9 of 45; 20%). During a median imaging follow-up of 7 months, 183 of 202 patients (90.1%) remained free of type Ia endoleaks. Primary prophylactic patients were free from type Ia endoleak in 110 of 114 cases (96.5%). The most challenging subset was revision patients treated for type Ia endoleak; type Ia endoleaks were evident during follow-up in 10 of 29 of the cases (34%). Sac regression >5 mm in patients with 1-year imaging was observed in 26 of 66 patients (39%) and was highest in the primary prophylaxis subset (20 of 43; 47%). CONCLUSIONS: EndoAnchor implantation can be a useful adjunct to EVAR as prophylaxis against proximal attachment site complications in patients with hostile aortic neck anatomy, as treatment for early and late type Ia endoleaks, or, in conjunction with aortic extension cuffs, for endograft migration. Whereas the most challenging patients are those who present with type Ia endoleaks remote from initial EVAR, EndoAnchors are still effective in treating the majority of these cases.

Results of the ANCHOR prospective, multicenter registry of EndoAnchors for type Ia endoleaks and endograft migration in patients with challenging anatomy.

OBJECTIVE: Proximal attachment site complications continue to occur after endovascular repair of abdominal aortic aneurysms (EVAR), specifically type Ia endoleak and endograft migration. EndoAnchors (Aptus Endosystems, Sunnyvale, Calif) were designed to enhance endograft proximal fixation and sealing, and the current study was undertaken to evaluate the potential benefit of this treatment. METHODS: During the 23-month period ending in December 2013, 319 subjects were enrolled at 43 sites in the United States and Europe. EndoAnchors were implanted in 242 patients (75.9%) at the time of an initial EVAR procedure (primary arm) and in 77 patients with an existing endograft and proximal aortic neck complications (revision arm). Technical success was defined as deployment of the desired number of EndoAnchors, adequate penetration of the vessel wall, and absence of EndoAnchor fracture. Procedural success was defined as technical success without a type Ia endoleak at completion angiography. Values are expressed as mean ± standard deviation and interquartile range. RESULTS: The 238 male (74.6%) and 81 female (25.4%) subjects had a mean age of 74.1 ± 8.2 years. Aneurysms averaged 58 ± 13 (51-63) mm in diameter at the time of EndoAnchor implantation (core laboratory measurements). The proximal aortic neck averaged 16 ± 13 (7-23) mm in length (42.7% <10 mm and 42.7% conical) and 27 ± 4 mm (25-30 mm) in diameter; infrarenal neck angulation was 24 ± 15 (13-34) degrees. The number of EndoAnchors deployed was 5.8 ± 2.1 (4-7). Technical success was achieved in 303 patients (95.0%) and procedural success in 279 patients (87.5%), 217 of 240 (89.7%) and 62 of 77 (80.5%) in the primary and revision arms, respectively. There were 29 residual type Ia endoleaks (9.1%) at the end of the procedure. During mean follow-up of 9.3 ± 4.7 months, 301 patients (94.4%) were free from secondary procedures. Among the 18 secondary procedures, eight were performed for residual type Ia endoleaks and the others were unrelated to EndoAnchors. There were no open surgical conversions, there were no aneurysm-related deaths, and no aneurysm ruptured during follow-up. CONCLUSIONS: Use of EndoAnchors to treat existing and acute type Ia endoleaks and endograft migration was successful in most cases. Prophylactic use of EndoAnchors in patients with hostile aortic neck anatomy appears promising, but definitive conclusions must await longer term follow-up data.

Synthesis and biological evaluation of substituted benzoxazoles as inhibitors of mPGES-1: use of a conformation-based hypothesis to facilitate compound design.

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) is a novel therapeutic target for the treatment of inflammation and pain. In the preceding letter, we detailed the discovery of clinical candidate PF-04693627, a potent mPGES-1 inhibitor possessing a novel benzoxazole structure. While PF-04693627 was undergoing further preclinical profiling, we sought to identify a back-up mPGES-1 inhibitor that differentiated itself from PF-04693627. The design, synthesis, mPGES-1 activity and in vivo PK of a novel set of substituted benzoxazoles are described herein. Also described is a conformation-based hypothesis for mPGES-1 activity based on the preferred conformation of the cyclohexane ring within this class of inhibitors.

Discovery and SAR of PF-4693627, a potent, selective and orally bioavailable mPGES-1 inhibitor for the potential treatment of inflammation.

Inhibition of mPGES-1, the terminal enzyme in the arachidonic acid/COX pathway to regulate the production of pro-inflammatory prostaglandin PGE2, is considered an attractive new therapeutic target for safe and effective anti-inflammatory drugs. The discovery of a novel series of orally active, selective benzoxazole piperidinecarboxamides as mPGES-1 inhibitors is described. Structure-activity optimization of lead 5 with cyclohexyl carbinols resulted in compound 12, which showed excellent in vitro potency and selectivity against COX-2, and reasonable pharmacokinetic properties. Further SAR studies of the benzoxazole ring substituents lead to a novel series of highly potent compounds with improved PK profile, including 23, 26, and 29, which were effective in a carrageenan-stimulated guinea pig air pouch model of inflammation. Based on its excellent in vitro and in vivo pharmacological, pharmacokinetic and safety profile and ease of synthesis, compound 26 (PF-4693627) was advanced to clinical studies.

Novel benzoxazole inhibitors of mPGES-1.

A novel series of potent benzoxazole mPGES-1 inhibitors has been derived from a hit from a high throughput screen. Compound 37 displays mPGES-1 inhibition in an enzyme assay (0.018 µM) and PGE-2 inhibition in a cell-based assay (0.034 µM). It demonstrates 500- and 2500-fold selectivity for mPGES-1 over COX-2 and 6-keto PGF-1α, respectively. In vivo PK studies in dogs demonstrate 55% oral bioavailability and an 7 h half-life.

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor.

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E(2) (PGE(2)) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE(2) synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC(50)=16.5+/-3.8nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE(2) synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE(2) synthesis (IC(50) in the range of 0.5-5 microM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF(1alpha) (PGF(1alpha)) and PGF(2alpha). In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE(2), PGF(1alpha) and PGF(2alpha) synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.

Secure fixation following EVAR with the Powerlink XL System in wide aortic necks: results of a prospective, multicenter trial.

PURPOSE: Endovascular stent graft repair of abdominal aortic aneurysms (AAA) with the Endologix Powerlink System (Endologix, Inc, Irvine, Calif) has been shown to be a safe and effective alternative to open surgery in patients having an aortic neck diameter of up to 26 mm. We assessed the safety and effectiveness of AAA repair in patients with wide aortic necks (up to 32 mm in diameter) using the Powerlink XL System. METHODS: Between September 2005 and June 2008, a prospective, multicenter, pivotal US Food and Drug Administration trial of the Powerlink XL System for endovascular aneurysm repair was conducted at 13 centers. Using a sizing algorithm based on computed tomography scan (CT)-based measurements, a total of 78 patients (N = 60 [pivotal trial]; N = 18 [continued access]) presenting with AAA and an infrarenal aortic neck up to 32 mm in diameter received a bifurcated stent graft via anatomical fixation at the aortoiliac bifurcation and proximal sealing with a Powerlink XL infrarenal proximal extension stent graft. Postoperatively, results were assessed with contrast-enhanced CT scans and abdominal x-rays at one, six, and 12 months, with continued annual follow-up to five years. RESULTS: Predominantly male (91%), patients presented at a mean age of 73 +/- 8.6 years with mean maximum aortic neck and AAA diameters of 31 +/- 1.9 mm (range, 25 to 32 mm) and 5.7 +/- 1.0 cm (range, 4.3 to 10 cm), respectively. Challenging infrarenal aortic neck anatomy, defined as the presence of severe thrombus and/or reverse taper, was present in 85% of patients. Technical success was achieved in 98.7% of patients, with one patient requiring femoral-femoral bypass intraoperatively. Aneurysm exclusion was achieved in 100% of patients over a mean procedure time of 129 +/- 66 minutes. Patients were discharged at a mean of 2.2 days postoperatively. At the one-month CT scan, the independent core lab identified a Type II endoleak in 13 patients, distal Type I and Type II endoleak in one patient, and unknown endoleak in three patients. At 30 days, there were no deaths, conversions, ruptures, or migrations. Through one year follow-up, Type II endoleak predominated (9/10 patients with endoleak), with one proximal Type I and no Type III, IV, or unknown endoleak; no conversions, ruptures, or migrations have been observed. The one-year all-cause mortality rate was 6.4%, with 100% freedom from aneurysm-related mortality. Secondary procedures were performed within one year in five patients (6.4%) for treatment of proximal Type I endoleak (n = 2), proximal Type I/Type II endoleak (n = 1), and distal Type I endoleak (n = 2). Reduced or stable aneurysm sac diameter at one year is observed in 96% of patients. CONCLUSIONS: The combination of an anatomically-fixed Powerlink bifurcated stent graft and a Powerlink XL infrarenal proximal extension appears safe and effectively excludes aneurysms in patients with wide aortic necks. These results suggest that fixation at the aortic bifurcation can provide secure fixation for patients with large diameter diseased proximal aortic necks.

Homo-timeric structural model of human microsomal prostaglandin E synthase-1 and characterization of its substrate/inhibitor binding interactions.

Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic pi-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H(2) (PGH(2)), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH(2) in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.