ENDOXY - Development of a Biomimetic Oxygenator-Test-Device

Objective

This study focusses on the development of a biomimetic oxygenator test device. Due to limited biocompatibility, current oxygenators do not allow mid- to long-term therapy. Tissue engineering uses autologous cell sources to overcome the immunogenic barriers of biomaterials. Surface coating with endothelial cells might improve hemocompatibility and thus prevent immunogenic reactions of the body. In this study this concept is applied to endothelialise a gas-permeable membrane to develop a biomimetic oxygenator test-device (ENDOXY).

Methods

ENDOXY—a multifunctional test-system was developed to endothelialise a gas-permeable membrane suitable for cell culture and to test the cell retention under shear stress and to measure gas transfer through it.

Results

Successful endothelialisation of the membrane was achieved and cells showed characteristic endothelial morphologies. They stained positive for endothelial markers. The number of cells aligned with shear stress and cell retention after blood perfusing experiments was high. Gas transfer is observed via uncoated and endothelialised membranes.

Conclusion

The study showed promising results with regard to system design, endothelialisation, and cell retention under shear stress conditions. It strongly encourages further research into the system by testing different membrane materials to design a biomimetic membrane surface and pave way for a fully hemocompatible oxygenator.     

Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167

To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram-positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected.     

Impact of the configuration of a chiral,activating carrier on the enantioselectivity of entrapped lipase from Candida rugosa in cyclohexane

Lipase from Candida rugosa was loaded into an amphiphilic polymer co-network (APCN) composed of the chiral poly[(R)-N-(1-hydroxybutan-2-yl) acrylamide] [P-(R)-HBA] and P-(S)-HBA, respectively, linked by poly(dimethylsiloxane). The nanophase-separated amphiphilic morphology affords a 38,000-fold activation of the enzyme in the esterification of 1-phenylethanol with vinyl acetate. Further, the enantioselectivity of the entrapped lipase was influenced by the configuration of the chiral, hydrophilic polymer matrix. While the APCN with the (S)-configuration of the APCN affords 5.4 faster conversion of the (R)-phenylethanol compared to the respective (S)-enantiomer, the (R)-APCN allows an only a 2.8 faster conversion of the (R)-enantiomer of the alcohol. Permeation-experiments reveal that the enantioselectivity of the reaction is at least partially caused by specific interactions between the substrates and the APCN.     

Grain Unloading of Arsenic Species in Rice

Rice (Oryza sativa) is the staple food for over half the world''s population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a ± stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.Paddy rice (Oryza sativa) is particularly effective, compared to other cereals, at accumulating arsenic (As) in shoot and grain (Williams et al., 2007b). Rice is the staple food for over half the world''s population (Fageria, 2007) and rice represents a significant dietary source of inorganic As, a class 1, nonthreshold carcinogen, particularly in Southeast Asia (Meharg et al., 2009). Inorganic As levels in rice grain are problematic even where soil As is at background levels, derived from geogenic sources (Lu et al., 2009; Meharg et al., 2009). However, widespread pollution of paddy soils with As, leading to further elevation of grain As, has occurred in some regions due to base and precious mining (Liao et al., 2005; Zhu et al., 2008), irrigation of paddies with As-elevated groundwaters (e.g. Meharg and Rahman, 2003; Williams et al., 2006), and the use of arsenical pesticides (Williams et al., 2007a). Unlike other cereal grains, paddy rice cultivation is dependent of soils being anaerobic, and it is this anoxia that gives rise to elevated As concentrations in the plant. Anaerobic soil conditions lead to the mobilization of As as arsenite, where under aerobic systems arsenate dominates (Xu et al., 2008). Arsenite is efficiently assimilated by rice roots through silicic acid transport pathway (Ma et al., 2008).Knowledge of As metabolism and partitioning within plants, particularly rice, is still developing rapidly (Zhao et al., 2009). Several studies have now shown that As in rice vegetative tissue and grain is predominantly speciated as inorganic As and the methylated species dimethylarsinic acid (DMA), with variable, though low, levels of monomethyl arsonic acid (MMA; Abedin et al., 2002a; Williams et al., 2005, 2006; Norton et al., 2009). Arsenate is an analog of phosphate and competes with phosphate for rice root uptake (Abedin et al., 2002a) while arsenite is taken up by rice roots via silicic acid transporters (Ma et al., 2008). Abedin et al. (2002b) demonstrated that the methylated species DMA and MMA are also taken up by rice plants although at a much slower rate than inorganic As, with the protonated neutral forms also transported through silicic acid pathway (Li et al., 2009). Arsenate is reduced to arsenite within the rice root (Xu et al., 2008; Zhao et al., 2009), which then enters the xylem via a silicic acid/arsenite effluxer (Ma et al., 2008; Zhao et al., 2009). Arsenite may be detoxified through complexation with thiol-rich peptides including phytochelatins (PCs) and glutathione followed by sequestration into vacuoles (Bleeker et al., 2006; Raab et al., 2007b; Zhao et al., 2009). Raab et al. (2007a) found that while methylated As species are taken up by rice roots much less efficiently than inorganic species, they appear to be translocated within the plant more efficiently. The comparative contributions of xylem and phloem transport, in translocation of As to the grain, are unknown.The main species within rice grain, along with DMA, are inorganic As, particularly arsenite, which may be complexed with thiols (Williams et al., 2005; Lombi et al., 2009). Nutrients are unloaded into the grain from the ovular vascular trace (OVT) into the nucellar tissue and from there are uploaded, via the apoplast into the filial tissue (the aleurone and the endosperm; Krishnan and Dayanandan, 2003). Lombi et al. (2009) recently suggested that this may represent a physiological barrier that As species cross with differential efficiency. However, the transport and unloading of As to/into the grain, which are key processes in terms of human exposure to this contaminant, are far from being fully understood.This study investigated the differential efficiency with which important As species are translocated and unloaded into the rice grain and the comparative contributions of phloem and xylem transport. Rice panicles were excised below the flag leaf node during grain development, 10 DPA, and treated to a hydroponically administered 48-h pulse of arsenite, arsenate, arsenite glutathione, or DMA. Total As concentrations in flag leaf, grain, and husk samples for each treatment were quantified by inductively coupled plasma mass spectroscopy (ICP-MS), and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy (XANES) analysis. To evaluate the contributions of phloem versus xylem transport, a stem-girdling treatment was applied, using steam to destroy phloem cells in a second set of panicles prior to a pulse of either DMA or arsenite. The spatial unloading of As species into the developing grain was examined by synchrotron x-ray fluorescence (XRF) mapping, and fluorescence microtomography for the DMA and arsenite treatments.     

Correlating Macro and Atomic Structure with Elastic Properties and Ionic Transport of Glassy Li2S‐P2S5 (LPS) Solid Electrolyte for Solid‐State Li Metal Batteries

A combination of high ionic conductivity and facile processing suggest that sulfide‐based materials are promising solid electrolytes that have the potential to enable Li metal batteries. Although the Li₂S‐P₂S₅ (LPS) family of compounds exhibit desirable characteristics, it is known that Li metal preferentially propagates through microstructural defects, such as particle boundaries and/or pores. Herein, it is demonstrated that a near theoretical density (98% relative density) LPS 75‐25 glassy electrolyte exhibiting high ionic conductivity can be achieved by optimizing the molding pressure and temperature. The optimal molding pressure reduces porosity and particle boundaries while preserving the preferred amorphous structure. Moreover, molecular rearrangements and favorable Li coordination environments for conduction are attained. Consequently, the Young's Modulus approximately doubles (30 GPa) and the ionic conductivity increases by a factor of five (1.1 mS cm^?1) compared to conventional room temperature molding conditions. It is believed that this study can provide mechanistic insight into processing‐structure‐property relationships that can be used as a guide to tune microstructural defects/properties that have been identified to have an effect on the maximum charging current that a solid electrolyte can withstand during cycling without short‐circuiting.     

Guanylyl cyclase‐G is an alarm pheromone receptor in mice

Many animals respond to threats by releasing alarm pheromones (APs) that warn conspecifics. In mice, detection of the AP 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT) is mediated by chemosensory neurons residing in the Grueneberg ganglion (GG) of the anterior nasal region. Although the molecular mechanisms underlying activation of GG neurons by SBT and other substances are still unclear, recent studies have reported an involvement of the transmembrane guanylyl cyclase (GC) subtype GC‐G in chemosensory signaling in the GG. Here, we show that SBT directly binds with high affinity to the extracellular domain of GC‐G and elicits an enhanced enzymatic activity of this protein. In line with this finding, heterologous expression of GC‐G renders cells responsive to SBT while activation by SBT was strongly attenuated in GG neurons from GC‐G‐deficient mice. Consistently, SBT‐induced fear‐associated behaviors, SBT‐evoked elevated blood pressure, and increased serum levels of the stress hormone corticosterone were clearly reduced in GC‐G‐knockout animals compared to wild‐type mice. These observations suggest that GC‐G serves as an unusual receptor in GG neurons mediating the detection of the volatile AP substance SBT.     

Pentoxifylline administration changes protein expression profile of coronary artery disease patients

Increased level of inflammatory mediators plays a central role in the features of coronary artery diseases. As pentoxifylline could suppress the inflammatory process and has shown some promising beneficial effects in inflammatory diseases, we evaluated the effect of two months pentoxifylline administration in proteome of PBMCs of patients with coronary artery disease (CAD). A randomized placebo-controlled study was used. Fourteen CAD patients were randomized to 2 months of pentoxifyline treatment (1200 mg/day) (n = 7) or placebo treatment (n = 7). Blood samples were obtained before and after treatment. A comparative 2 dimensional gel electrophoresis was performed, and gels were silver-stained. Differentially expressed protein spots were detected and were identified by MALDI-TOF spectrometry. Six differentially expressed proteins were identified as HSP70, PPIA and α-Enolase, (all up-regulated) S100-A9, PIMT and β-5 tubulin (all down-regulated), most of which had previously been shown to play a potential role in the pathogenesis of atherosclerosis. As the blood mononuclear cell proteome responds to pentoxifylline with changes in a number of atherosclerosis-relevant proteins, it seems that pentoxifylline could be a good choice for future studies for prevention of cardiovascular events.     

Characterization of the eugenol hydroxylase genes (ehyA/ehyB) from the new eugenol-degrading Pseudomonas sp. strain OPS1

During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for an eugenol dehydrogenase of P fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.     

Identification of chronic heart failure patients with a high 12-month mortality risk using biomarkers including plasma C-terminal pro-endothelin-1

Objectives

We hypothesised that assessment of plasma C-terminal pro-endothelin-1 (CT-proET-1), a stable endothelin-1 precursor fragment, is of prognostic value in patients with chronic heart failure (CHF), beyond other prognosticators, including N-terminal pro-B-type natriuretic peptide (NT-proBNP).

Methods

We examined 491 patients with systolic CHF (age: 63±11 years, 91% men, New York Heart Association [NYHA] class [I/II/III/IV]: 9%/45%/38%/8%, 69% ischemic etiology). Plasma CT-proET-1 was detected using a chemiluminescence immunoassay.

Results

Increasing CT-proET-1 was a predictor of increased cardiovascular mortality at 12-months of follow-up (standardized hazard ratio 1.42, 95% confidence interval [CI] 1.04–1.95, p = 0.03) after adjusting for NT-proBNP, left ventricular ejection fraction (LVEF), age, creatinine, NYHA class. In receiver operating characteristic curve analysis, areas under curve for 12-month follow-up were similar for CT-proET-1 and NT-proBNP (p = 0.40). Both NT-proBNP and CT-proET-1 added prognostic value to a base model that included LVEF, age, creatinine, and NYHA class. Adding CT-proET-1 to the base model had stronger prognostic power (p<0.01) than adding NT-proBNP (p<0.01). Adding CT-proET-1 to NT-proBNP in this model yielded further prognostic information (p = 0.02).

Conclusions

Plasma CT-proET-1 constitutes a novel predictor of increased 12-month cardiovascular mortality in patients with CHF. High CT-proET-1 together with high NT-proBNP enable to identify patients with CHF and particularly unfavourable outcomes.