Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-D-glutamate capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded and the immune system can successfully combat the infection. While CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improves production properties and dramatically increased kinetic thermostability: At 45(o)C CapD is completely inactive after 5 minutes, but circularly permuted CapD remains almost entirely active after 30 minutes. In addition, we identify an amino acid substitution that dramatically decreases transpeptidation activity, but not hydrolysis. Subsequently, we show that this mutant has a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum, rather than hydrolysis.

The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.

A review of the issues involved in placement of Tunneled Dialysis Catheter with a suggested scheme for maximizing good catheter placement and function.

Background: Hemodialysis (HD) grafts often fail due to stenosis at the venous anastomosis and thrombotic occlusion. Percutaneous management relies on thrombolysis with plasminogen activators, mechanical removal of thrombus and angioplasty of the stenotic lesion. Objectives: This report describes a phase I trial using Plasmin (Human) TAL 05-00018, a direct-acting fibrinolytic agent, to evaluate safety and, secondarily, to establish effective thrombolytic dosing. Patients/methods: Six cohorts of 5 patients with acute HD graft occlusion documented by angiography were treated with escalating dosages of plasmin (1, 2, 4, 8, 12, and 24 mg) infused over 30 minutes via criss-crossed pulse-spray catheters within the graft. The primary efficacy endpoint was >/=50% thrombolysis, as determined by comparison of pre- and 30-minute post-plasmin fistulograms. Results: Of 31 subjects who received study drug (safety population), 1 withdrew and 30 completed the trial (evaluable for efficacy). There was no significant change in plasma alpha-2 antiplasmin or fibrinogen concentration, major bleeding did not occur and there were no deaths. Serious adverse events in 4 patients were not related to study drug. There was a dose-response relationship for the primary efficacy endpoint, all 5 subjects receiving 24 mg achieving >75% lysis. Conclusions: This first phase I study of Plasmin (Human) TAL 05-00018, infused into thrombosed HD grafts, documents safety at dosages of 1 to 24 mg and an effective thrombolytic dosage of 24 mg. The results establish a foundation for further clinical study of catheter-based plasmin administration in thrombotic disorders.

PURPOSE: To compare the effectiveness and safety of use of the peripheral cutting balloon (PCB) versus standard percutaneous transluminal angioplasty (PTA) for the treatment of hemodialysis-related stenoses. MATERIALS AND METHODS: This prospective, randomized multicenter clinical trial included 340 patients with stenotic or thrombosed hemodialysis grafts who were randomized to receive treatment with the PCB or PTA for venous outflow stenosis. One hundred seventy-three patients underwent treatment with the PCB, 101 with stenotic grafts and 72 with thrombosed grafts. PTA was used to treat 167 patients, 94 patients with stenotic grafts and 73 with thrombosed grafts. The follow-up period extended for 6 months. RESULTS: The procedural success rates were 80.8% and 75.4% for the PCB and PTA groups, respectively (P =.24). With use of the PCB, the primary patency rates of the target lesions were 84.3%, 65.8%, and 47.9% at 1 month, 3 months, and 6 months, respectively. With PTA, the primary patency rates of the target lesions were 77.7%, 63.4%, and 40.5% at 1 month, 3 months, and 6 months, respectively. The primary patency rates of the entire vascular access circuit were 82.6%, 61.0%, and 43.3% at 1 month, 3 months, and 6 months, respectively, with use of the PCB. For patients who were treated with PTA, the primary patency rates of the vascular access circuit were 75.9%, 61.0%, and 36.3% at 1 month, 3 months, and 6 months, respectively. When comparing the PCB and PTA, there was no difference in the 6-month primary patency rates in the target lesion (P =.373) or the entire vascular access circuit (P =.531). There were nine device-related complications in the PCB group (5.2%): five venous ruptures (2.9%), three venous dissections (1.7%), and one case of thrombosis (0.6%). There were no device-related complications in the PTA group. CONCLUSION: This prospective, randomized trial comparing use of the PCB versus standard PTA for treatment of hemodialysis-related venous stenoses demonstrated that the PCB provides equivalent 6-month patency to PTA for stenotic and thrombosed grafts.

P:URPOSE: To evaluate the safety and efficacy of a hydrodynamic thrombectomy system in a prospective, multicenter randomized comparison with pulse-spray thrombolysis in hemodialysis grafts. MATERIALS AND METHODS: Nine centers enrolled 120 adult patients with recently (</=14 days) thrombosed hemodialysis grafts. Graft venography was used to confirm occlusion in 62 patients randomly assigned to thrombectomy and 58 to thrombolysis. For thrombolysis, a mixture of 5,000 U of heparin and 250,000 U of urokinase was distributed throughout the thrombus, first to the venous then to the arterial graft end. For thrombectomy, the catheter was passed in the same sequence. Technical success was removal of 80% or more of thrombus. Clinical success was technical success plus the ability to dialyze. Also assessed were total procedure time, thrombus treatment time, procedure-related blood loss, other complications, and 30- and 90-day outcomes. RESULTS: Patient demographics were comparable. Technical success rates were 95%(59 of 62) for thrombectomy and 90%(52 of 58) for thrombolysis (P:=.31). Clinical success rates were 89%(55 of 62) and 81%(47 of 58), respectively (P:=.24). At 30 days, 69%(43 of 62) and 66%(38 of 58), respectively, could be dialyzed through the graft (P:=.70); at 90 days, the rates were 40%(25 of 62) and 41%(24 of 58), respectively (P:=.91). None of these differences or those for procedure-related blood loss and early and late complications were statistically significant. Thrombus treatment times of 16.8 minutes for thrombectomy and 23.4 minutes for thrombolysis were significantly different (P:<.01). CONCLUSION: The hydrodynamic thrombectomy system is at least as efficacious and safe as pulse-spray thrombolysis but shortens thrombus treatment time.

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.