The evolution of an ideal stent design and its impact on the aortic endothelium during and after percutaneous replacement

Vascular support structures are important devices for treating valve stenosis. A large population of patients is treated for valvular disease and the preferred mode of treatment is percutaneous valve replacement. Stent devices are proving to be an improved technology in minimally invasive cardiac surgery. This new technology provides highly effective results at minimal cost and with a short duration of hospitalisation. Stents as a supporting structure for tissue valves have evolved over the years into remarkably useful and effective devices. During this process, a number of specific designs have come and gone, and a few have remained. Many design changes were successful, and many were not. This article describes the merits and demerits of various stent designs and details the specific reasons why a particular novel design is expected to be the most suitable implant during and after percutaneous aortic valve replacement.     

Dimethyl Disulfide Produced by the Naturally Associated Bacterium Bacillus sp B55 Promotes Nicotiana attenuata Growth by Enhancing Sulfur Nutrition

Bacillus sp B55, a bacterium naturally associated with Nicotiana attenuata roots, promotes growth and survival of wild-type and, particularly, ethylene (ET)–insensitive 35S-ethylene response1 (etr1) N. attenuata plants, which heterologously express the mutant Arabidopsis thaliana receptor ETR1-1. We found that the volatile organic compound (VOC) blend emitted by B55 promotes seedling growth, which is dominated by the S-containing compound dimethyl disulfide (DMDS). DMDS was depleted from the headspace during cocultivation with seedlings in bipartite Petri dishes, and ³⁵S was assimilated from the bacterial VOC bouquet and incorporated into plant proteins. In wild-type and 35S-etr1 seedlings grown under different sulfate (SO₄⁻²) supply conditions, exposure to synthetic DMDS led to genotype-dependent plant growth promotion effects. For the wild type, only S-starved seedlings benefited from DMDS exposure. By contrast, growth of 35S-etr1 seedlings, which we demonstrate to have an unregulated S metabolism, increased at all SO₄⁻² supply rates. Exposure to B55 VOCs and DMDS rescued many of the growth phenotypes exhibited by ET-insensitive plants, including the lack of root hairs, poor lateral root growth, and low chlorophyll content. DMDS supplementation significantly reduced the expression of S assimilation genes, as well as Met biosynthesis and recycling. We conclude that DMDS by B55 production is a plant growth promotion mechanism that likely enhances the availability of reduced S, which is particularly beneficial for wild-type plants growing in S-deficient soils and for 35S-etr1 plants due to their impaired S uptake/assimilation/metabolism.     

Taxonomy of the ‘Synargis axenus complex’ belonging to the ‘Synargis regulus’ species group,with a phylogenetic reassessment of the genus Synargis Hübner, 1819 (Lepidoptera: Riodinidae: Nymphidiini)

Three new species of Synargis Hübner, 1819, from Paraguay and southern and central Brazil are described: Synargis fandanga sp. nov. from Paraguay (Amambay and Paraguari) and southern Brazil (Paraná and Santa Catarina), Synargis rasqueada sp. nov. from central Brazil (Mato Grosso), and Synargis gorpa sp. nov. from southern Brazil (Paraná, Santa Catarina, and Rio Grande do Sul). Lectotypes are designated for Lemonias axenus Hewitson, 1876, Ematurgina axenus ochrophlegma Sitchel, 1911, Ematurgina acervata Seitz, 1932, and Ematurgina perrupta Seitz, 1932. Ematurgina ochrophlegma f. dissimilis Hayward, 1949, is a new synonym of Synargis bifasciata (Mengel, 1902), and Ematurgina ochrophlegma f. distincta Hayward, 1949, is a new synonym of Synargis axenus (Hewitson, 1876). The revalidation of E. perrupta Seitz, 1932, and the new status Synargis ochrophlegma (Stichel, 1911) are proposed. Ematurgina perrupta ab. roeberi Seitz, 1932, and Ematurgina bifasciata ochrophlegma ab. leucomelaina Breyer, 1930, are considered unavailable names. Based on a previous phylogenetic hypothesis, the phylogeny of the genus Synargis is reassessed, adding these new and revalidated taxa, and nine additional characters. The ‘Synargis regulus’ species group and the ‘Synargis axenus complex’ are recovered as monophyletic, with S. gorpa sp. nov. sister to the remaining species of the ‘S. axenus complex’. Additionally, an up‐to‐date geographical distribution map and a dichotomous key are provided, and the taxonomy of the taxa involved is discussed. © 2013 The Linnean Society of London     

Synthesis and properties of fatty acid starch esters

Being completely bio-based, fatty acid starch esters (FASEs) are attractive materials that represent an alternative to crude oil-based plastics. In this study, two synthesis methods were compared in terms of their efficiency, toxicity and, especially, product solubility with starch laurate (C₁₂) as model compound. Laurates (DS > 2) were obtained through transesterification of fatty acid vinylesters in DMSO or reaction with fatty acid chlorides in pyridine. The latter lead to higher DS-values in a shorter reaction time. But due to the much better solubility of the products compared to lauroyl chloride esterified ones, vinylester-transesterification was preferred to optimize reaction parameters, where reaction time could be shortened to 2 h. FASEs C₆–C₁₈ were also successfully prepared via transesterification. To determine the DS of the resulting starch laurates, the efficient ATR-IR method was compared with common methods (elementary analysis, ¹H NMR). Molar masses (M_w) of the highly soluble starch laurates were analyzed using SEC-MALLS (THF). High recovery rates (>80%) attest to the outstanding solubility of products obtained through transesterification, caused by a slight disintegration during synthesis. Particle size distributions (DLS) demonstrated stable dissolutions in CHCl₃ of vinyl laurate esterified – contrary to lauroyl chloride esterified starch. For all highly soluble FASEs (C₆–C₁₈), formation of concentrated solutions (10 wt%) is feasible.     

Desensitization and Internalization of Endothelin Receptor A: IMPACT OF G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2)-MEDIATED PHOSPHORYLATION*

Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.     

Computational biomechanics of a lumbar motion segment in pure and combined shear loads

Anterior shear has been implicated as a risk factor in spinal injuries. A 3D nonlinear poroelastic finite element model study of a lumbar motion segment L4-L5 was performed to predict the temporal shear response under various single and combined shear loads. Effects of nucleotomy and facetectomy as well as changes in the posture and facet gap distance were analyzed as well.     

Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.The secretory expression of recombinant proteins can offer significant process advantages over cytosolic production strategies, since secretion into the growth medium greatly facilitates downstream processing and therefore can significantly reduce the costs of producing a desired target protein (Quax, 1997). And, in fact, the enormous secretion capacity of certain Gram-positive bacteria (e.g. various Bacillus species) has been used since many years in industry for the production of mainly host-derived secretory proteins such as proteases and amylases, resulting in amounts of more than 20 g l⁻¹ culture medium (Harwood and Cranenburg, 2008). In contrast, attempts to use Bacillus species for the secretory production of heterologous proteins have often failed or led to disappointing results, a fact that, among other reasons, could in many cases be attributed to the presence of multiple cell wall-associated and secreted proteases that rapidly degraded the heterologous target proteins (Li et al., 2004; Sarvas et al., 2004; Westers et al., 2011). Therefore, an increasing need exists to explore alternative host systems with respect to their ability to express and secrete problematic and/or complex heterologous proteins of biotechnological interest.So far, the Gram-positive bacterium Corynebacterium glutamicum has been used in industry mainly for the production of amino acids and other low-molecular weight compounds (Leuchtenberger et al., 2005; Becker and Wittmann, 2011; Litsanov et al., 2012). However, various recent reports have indicated that C. glutamicum might likewise possess a great potential as an alternative host system for the secretory expression of foreign proteins. Corynebacterium glutamicum belongs to a class of diderm Gram-positive bacteria that, besides the cytoplasmic membrane, possess an additional mycolic acid-containing outer membrane-like structure that acts as an extremely efficient permeability barrier for hydrophilic compounds (Hoffmann et al., 2008; Zuber et al., 2008). Despite this fact, an efficient secretion of various heterologous proteins into the growth medium of this microorganism has been observed (e.g. Billman-Jacobe et al., 1995; Meissner et al., 2007; Kikuchi et al., 2009; Tateno et al., 2009; Tsuchidate et al., 2011).In bacteria, two major export pathways exist for the transport of proteins across the cytoplasmic membrane that fundamentally differ with respect to the folding status of their respective substrate proteins during the actual translocation step. The general secretion (Sec) system transports its substrates in a more or less unfolded state and folding takes places on the trans side of the membrane after the actual transport event (Yuan et al., 2010; du Plessis et al., 2011). In contrast, the alternative twin-arginine translocation (Tat) system translocates its substrates in a fully folded form and therefore provides an attractive alternative for the secretory production of proteins that cannot be produced in a functional form via the Sec route (Brüser, 2007). Carbohydrate oxidases are biotechnologically interesting enzymes (van Hellemond et al., 2006) that are excluded from Sec-dependent secretion since they depend on a tightly or covalently bound cofactor for their activity and, for this reason, require that their folding and cofactor insertion has to take place in the cytosol. Because C. glutamicum has shown to be an excellent host for the Tat-dependent secretion of the cofactor-less model protein GFP (Meissner et al., 2007; Teramoto et al., 2011), we now asked whether it is likewise possible to secrete a cofactor-containing enzyme into the supernatant of C. glutamicum using the same protein export route.As a model protein, we chose the sorbitol–xylitol oxidase (SoXy) from Streptomyces coelicolor, a normally cytosolic enzyme that possesses a covalently bound FAD molecule as cofactor (Heuts et al., 2007; Forneris et al., 2008). FAD is incorporated into the apoprotein in a post-translational and self-catalytic process that only occurs if the polypeptide chain has adopted a correctly folded structure (Heuts et al., 2007; 2009). To direct SoXy into the Tat export pathway of C. glutamicum, we constructed a gene encoding a TorA–SoXy hybrid precursor in which SoXy is fused to the strictly Tat-specific signal peptide of the periplasmic Escherichia coli Tat substrate trimethylamine N-oxide reductase (TorA) (Fig. 1) which, in our previous study, has been proven to be a functional and strictly Tat-specific signal peptide also in C. glutamicum (Meissner et al., 2007). The corresponding torA–soxy gene was cloned into the expression vector pEKEx2 (Eikmanns et al., 1991) under the control of an IPTG-inducible P_tac promotor. After transformation of the resulting plasmid pTorA–SoXy into the C. glutamicum ATCC13032 wild-type strain, two independent colonies of the resulting recombinant C. glutamicum (pTorA–SoXy) strain and, as a control, a colony of a strain that contained the empty expression vector without insert [C. glutamicum (pEKEx2)] were grown in CGXII medium (Keilhauer et al., 1993) at 30°C for 16 h in the presence of 1 mM IPTG. Subsequently, the proteins present in the culture supernatants were analysed by SDS-PAGE followed by staining with Coomassie blue. As shown in Fig. 2, in the supernatants of the pTorA–SoXy-containing cells (lanes 3 and 4), a prominent protein band of approximately 44 kDa can be detected, the size of which is very similar to the calculated molecular mass (44.4 kDa) of SoXy. Since this band is completely lacking in the supernatant of the control strain (lane 2), this strongly suggests that this band corresponds to SoXy that has been secreted into the culture supernatant of C. glutamicum (pTorA–SoXy). And, in fact, this suggestion was subsequently confirmed in a direct way by MALDI-TOF mass spectrometry after extraction of the protein out of the gel followed by tryptic digestion (Schaffer et al., 2001) (data not shown).Open in a separate windowFigure 1The TorA–SoXy hybrid precursor protein. Upper part: Schematic drawing of the relevant part of the pTorA–SoXy expression vector. P_tac, IPTG-inducible tac promotor. RBS, ribosome binding site. To maintain the authentic TorA signal peptidase cleavage site, the first four amino acids of the mature TorA protein (black bar) were retained in the TorA–SoXy fusion protein. White bar: TorA signal peptide (TorA^SP); grey bar: SoXy (amino acids 2–418). Lower part: Amino acid sequence of the signal peptide and early mature region of the TorA–SoXy hybrid precursor. The twin-arginine consensus motif of the TorA signal peptide is underlined. The four amino acids derived from mature TorA are shown in italics. The signal peptidase cleavage site is indicated by an arrowhead.Open in a separate windowFigure 2Secretion of SoXy into the growth medium of C. glutamicum. Cells of C. glutamicum ATCC13032 containing the empty vector pEKEx2 and two independently transformed colonies of C. glutamicum (pTorA–SoXy) were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with CGXII medium (Keilhauer et al., 1993) and inoculated to an OD₆₀₀ of 0.5 in 5 ml of fresh CGXII medium containing 1 mM IPTG. After 16 h of further growth at 30°C, the supernatant fractions were prepared as described previously (Meissner et al., 2007). Samples corresponding to an equal number of cells were subjected to SDS-PAGE followed by staining with Coomassie blue. Lane 1, molecular mass marker (kDa). Lane 2, C. glutamicum (pEKEx2); lanes 3 and 4, C. glutamicum (pTorA–SoXy). The position of the secreted SoXy protein is indicated by an arrow.Next, the supernatant of C. glutamicum (pTorA–SoXy) was analysed for SoXy enzyme activity by measuring the production of H₂O₂ that is formed during the enzymatic conversion of sorbitol to fructose (Meiattini, 1983). Six hours after induction of gene expression by 1 mM IPTG, an enzymatic activity of 10.3 ± 1.6 nmol min⁻¹ ml⁻¹ could be determined in the supernatant of C. glutamicum (pTorA–SoXy). In contrast, no such activity was found in the supernatant of the control strain C. glutamicum (pEKEx2). From these results we conclude that we have succeeded in the secretion of enzymatically active and therefore FAD cofactor-containing SoXy into the culture supernatant of C. glutamicum.Finally, we examined whether the secretion of SoXy had in fact occurred via the Tat pathway of C. glutamicum. Plasmid pTorA–SoXy was used to transform C. glutamcium ATCC13032 wild type and a C. glutamicum ΔTatAC mutant strain that lacks two essential components of the Tat transport machinery and therefore does not possess a functional Tat translocase (Meissner et al., 2007). The corresponding cells were grown in BHI medium (Difco) at 30°C in the presence of 1 mM IPTG for 6 h. Subsequently, the proteins present in the cellular and the supernatant fractions of the corresponding cells were analysed by SDS-PAGE followed by Western blotting using SoXy-specific antibodies. As shown in Fig. 3, polypeptides corresponding to the unprocessed TorA–SoXy precursor and some minor smaller degradation products of it can be detected in the cellular fractions of both the wild-type and the ΔTatAC deletion strains (lanes 3 and 5). In the supernatant fraction of the Tat⁺ wild-type strain (lane 4), but not that of the ΔTatAC strain (lane 6), a polypeptide corresponding to mature SoXy is present, clearly showing that export of SoXy in the wild-type strain had occurred in a strictly Tat-dependent manner. Another noteworthy finding is the observation that hardly any mature SoXy protein accumulated in the cellular fraction of the Tat⁺ wild-type strain (lane 3), indicating that SoXy is, after its Tat-dependent translocation across the cytoplasmic membrane and processing by signal peptidase, rapidly transported out of the intermembrane space across the mycolic acid-containing outer membrane into the supernatant. However, the mechanism of how proteins cross this additional permeability barrier is completely unknown so far (Bitter et al., 2009).Open in a separate windowFigure 3Transport of TorA–SoXy occurs in a strictly Tat-dependent manner. Plasmid pTorA–SoXy was transformed into C. glutamcium ATCC13032 (Tat⁺) and a C. glutamicum ΔTatAC mutant that lacks a functional Tat translocase (Meissner et al., 2007). As a control, the empty pEKEx2 expression vector was transformed into C. glutamicum ATCC13032 (Tat⁺). The respective strains were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with BHI and resuspended in 20 ml of fresh BHI medium containing 1 mM IPTG. After 6 h of further growth at 30°C, the cellular (C) and supernatant (S) fractions were prepared as described previously (Meissner et al., 2007). Samples of the C and S fractions were subjected to SDS-PAGE followed by immunoblotting using anti-SoXy antibodies as indicated at the top of the figure. Lanes 1 and 2: C. glutamicum ATCC13032 (pEKEx2); lanes 3 and 4: C. glutamicum ATCC13032 (pTorA–SoXy); lanes 5 and 6: C. glutamicum ΔTatAC (pTorA–SoXy). Asterisk: TorA–SoXy precursor; arrow: secreted SoXy protein. The positions of molecular mass markers (kDa) are indicated at the left margin of the figure.To the best of our knowledge, our results represent the first documented example of the successful secretion of a normally cytosolic, cofactor-containing protein via the Tat pathway in an active form into the culture supernatant of a recombinant expression host. Our results clearly show that, also for this biotechnologically very interesting class of proteins, a secretory production strategy can be a promising alternative to conventional intracellular expression strategies. Besides for SoXy and other FAD-containing carbohydrate oxidases, for which various applications are perceived by industry such as the in situ generation of hydrogen peroxide for bleaching and disinfection performance in technical applications, their use in the food and drink industry, as well as their use in diagnostic applications and carbohydrate biosynthesis processes (Oda and Hiraga, 1998; Murooka and Yamashita, 2001; van Hellemond et al., 2006; Heuts et al., 2007), a secretory production strategy might now be an attractive option also for biotechnologically relevant enzymes that are used as biocatalysts in chemo-enzymatic syntheses and that possess cofactors other than FAD, such as pyridoxal-5′-phosphate (PLP)-dependent ω-transaminases (Mathew and Yun, 2012) or various thiamin diphosphate (TDP)-dependent enzymes (Müller et al., 2009).