Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.     

Structural Basis of Cyclic 1,3-Diene Forming Acyl-Coenzyme A Dehydrogenases

The biologically important, FAD-containing acyl-coenzyme A (CoA) dehydrogenases (ACAD) usually catalyze the anti-1,2-elimination of a proton and a hydride of aliphatic CoA thioesters. Here, we report on the structure and function of an ACAD from anaerobic bacteria catalyzing the unprecedented 1,4-elimination at C3 and C6 of cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA) to cyclohex-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) and at C3 and C4 of the latter to benzoyl-CoA. Based on high-resolution Ch1CoA dehydrogenase crystal structures, the unorthodox reactivity is explained by the presence of a catalytic aspartate base (D91) at C3, and by eliminating the catalytic glutamate base at C1. Moreover, C6 of Ch1CoA and C4 of Ch1,5CoA are positioned towards FAD-N5 to favor the biologically relevant C3,C6- over the C3,C4-dehydrogenation activity. The C1,C2-dehydrogenation activity was regained by structure-inspired amino acid exchanges. The results provide the structural rationale for the extended catalytic repertoire of ACADs and offer previously unknown biocatalytic options for the synthesis of cyclic 1,3-diene building blocks.     

Perceptual categories for spatial layout

The central problems of vision are often divided into object identification and localization. Object identification, at least at fine levels of discrimination, may require the application of top-down knowledge to resolve ambiguous image information. Utilizing top-down knowledge, however, may require the initial rapid access of abstract object categories based on low-level image cues. Does object localization require a different set of operating principles than object identification or is category determination also part of the perception of depth and spatial layout? Three-dimensional graphics movies of objects and their cast shadows are used to argue that identifying perceptual categories is important for determining the relative depths of objects. Processes that can identify the causal class (e.g. the kind of material) that generates the image data can provide information to determine the spatial relationships between surfaces. Changes in the blurriness of an edge may be characteristically associated with shadows caused by relative motion between two surfaces. The early identification of abstract events such as moving object/shadow pairs may also be important for depth from shadows. Knowledge of how correlated motion in the image relates to an object and its shadow may provide a reliable cue to access such event categories.     

The perils of permanent punctal plugs

Satisfactory pain relief with postoperative extradural bupivacaine increases the amount of breast feeding after Caesarean section. To investigate the effect of extradural buprenorphine, we have evaluated the amount of breast feeding and the gain in infant weight for 11 days after Caesarean section in patients who received continuous extradural bupivacaine with or without buprenorphine. Extradural buprenorphine significantly decreased both measures although there was no significant difference in pain intensity. We suggest that extradural buprenorphine suppressed breast feeding after Caesarean section.     

Isoflurane and halothane do not alter the enhanced afterload sensitivity of left ventricular relaxation in dogs with pacing-induced cardiomyopathy

BACKGROUND: The afterload dependence of left ventricular (LV) relaxation is accentuated in the failing heart. The authors tested the hypothesis that isoflurane and halothane alter the afterload sensitivity of LV relaxation in dogs with pacing-induced cardiomyopathy. METHODS: Dogs (n = 6) were chronically instrumented for measurement of LV and aortic pressures and subendocardial segment length. Hemodynamics were recorded, and LV relaxation was evaluated with a time constant of isovolumic relaxation (tau) under control conditions and during decreases and increases in LV load produced by abrupt inferior vena caval (IVC) occlusion and phenylephrine (intravenous infusion), respectively, in the conscious state and during isoflurane and halothane anesthesia (1.5 MAC) on separate days before and after the development of pacing-induced cardiomyopathy. The slope (R) of the tau versus LV end-systolic pressure (P[es]) relation was also used to determine the afterload sensitivity of LV relaxation. RESULTS: IVC occlusion and phenylephrine produced similar or less profound changes in P(es), regional end-systolic force (an index of LV afterload), and end-systolic segment length in cardiomyopathic compared with healthy dogs. However, IVC occlusion and phenylephrine caused more pronounced alterations in tau in conscious and isoflurane- and halothane-anesthetized dogs after the development of cardiomyopathy. R was also greater in cardiomyopathic compared with healthy dogs (e.g., 0.32 +/- 0.03 before pacing to 1.00 +/- 0.13 ms/mmHg in conscious dogs). No differences in the load dependence of LV relaxation were observed between the conscious and anesthetized states before and after production of LV dysfunction. CONCLUSIONS: The results indicate that isoflurane and halothane do not alter the afterload dependence of LV relaxation in the normal and cardiomyopathic heart. The lack of effect of the volatile anesthetics is probably related to anesthetic-induced reductions in the resistance to LV ejection concomitant with simultaneous negative inotropic effects.     

Simultaneous feature selection and Gaussian mixture model estimation for supervised classification problems

A new expectation maximization (EM) algorithm for time-critical supervised classification tasks in remote sensing is proposed. Compared to standard EM and other approaches, it has the following advantages: (1) No knowledge about the class distributions is needed. (2) The number of components is estimated. (3) It does not require careful initialization. (4) Singular estimates are avoided due to the ability of pruning components. (5) The best discriminating features are identified simultaneously. (6) The features are identified by incorporating Mahalanobis distances.     

Concession-making in multi-attribute auctions and multi-bilateral negotiations: Theory and experiments

Concession-making behavior is an essential process in negotiations and auctions and has critical impact on the outcomes of an exchange. In auctions, concessions relate to deciding on the next bid by bid-makers, while in negotiations they involve proposing next offers by both parties. The purpose of this paper is twofold: (1) present a theory of concessions which could be applied to both auctions and negotiations and (2) provide experimental verification of the theory. The concession model identifies nine types of concessions derived from the preference structures. The occurrence of all nine types has been confirmed in experiments. The model also identifies two concession categories which are associated with the process transparency. The theory-based assessment of the concession-making in multi-attribute auctions and multi-issue multi-bilateral negotiations allows for their comparison. To this end, two systems implemented on the Invite e-negotiation platform are used. One of the major findings suggests that auctions induce concession-making that is more likely to lead to Pareto-optimal agreements. This study also suggests that greater transparency in negotiations is likely to lead to better agreements.     

Self‐Immobilizing Oxidoreductases for Flow Biocatalysis in Miniaturized Packed‐Bed Reactors

The industrial implementation of enzymes in flow biocatalysis microreactors is expected to be essential for the emergence of a bio‐based circular economy. Major challenges concern the efficient immobilization of delicate enzymes inside miniaturized reactors without compromising their catalytic activity. We describe the exploitation of the widely used His‐tag system in a microfluidic packed‐bed reactor that contains ketoreductase‐functionalized magnetic beads. In a continuous process, these reactors produced highly stereoselective (R)‐configured alcohols (d.r. 99:1) with an average conversion of > 90 % for more than 4 days. We believe that such miniaturized flow reactors can be of great utility for future sustainable production processes.     

Evaporation of pyrolysis oil: Product distribution and residue char analysis

The evaporation of pyrolysis oil was studied at varying heating rates (～1–10⁶°C/min) with surrounding temperatures up to 850°C. A total product distribution (gas, vapor, and char) was measured using two atomizers with different droplet sizes. It was shown that with very high heating rates (～10⁶°C/min) the amount of char was significantly lowered (～8%, carbon basis) compared to the maximum amount, which was produced at low heating rates using a TGA (～30%, carbon basis; heating rate 1°C/min). The char formation takes place in the 100–350°C liquid temperature range due to polymerization reactions of compounds in the pyrolysis oil. All pyrolysis oil fractions (whole oil, pyrolytic lignin, glucose and aqueous rich/lean phase) showed charring behavior. The pyrolysis oil chars age when subjected to elevated temperatures (≥700°C), show similar reactivity toward combustion and steam gasification compared with chars produced during fast pyrolysis of solid biomass. However, the structure is totally different where the pyrolysis oil char is very light and fluffy. To use the produced char in conversion processes (energy or syngas production), it will have to be anchored to a carrier. © 2010 American Institute of Chemical Engineers AIChE J, 2010     

Cover Picture: Screening for Cytochrome P450 Reactivity by Harnessing Catalase as Reporter Enzyme (ChemBioChem 4/2009)

The cover picture shows a bacterial cytochrome P450 enzyme (CYP152A1, blue protein) screening for new substrates, such as nifidepine (highlighted green). The identification of novel reactivities of P450 enzymes is of major importance for applications in biocatalysis, biosensing and metabolic engineering. In their contribution on p. 751 ff, Niemeyer et al. report a novel assay for the rapid and facile screening of substrate libraries for organic hydroperoxide‐mediated P450 reactivity. Peroxide depletion is monitored in a fluorescence microplate assay, by harnessing a previously undescribed reactivity of the enzyme catalase (orange protein structure). The assay thus connects the occurrence of P450 reactivity with a universal read‐out, thereby circumventing the need for substrate‐specific detection schemes.