High efficiency ethanol fermentation by entrapment of Zymomonas mobilis into LentiKats

AIMS: To examine the potential of Zymomonas mobilis entrapped into polyvinylalcohol (PVA) lens-shaped immobilizates in batch and continuous ethanol production. METHODS AND RESULTS: Cells, free or immobilized in PVA hydrogel-based lens-shaped immobilizates - LentiKats, were cultivated on glucose medium in a 1 l bioreactor. In comparison with free cell cultivation, volumetric productivity of immobilized batch culture was nine times higher (43.6 g l(-1) h(-1)). The continuously operated system did not improve the efficiency (volumetric productivity of the immobilized cells 30.7 g l(-1) h(-1)). CONCLUSIONS: We demonstrated Z. mobilis capability, entrapped into LentiKats, in the cost-efficient batch system of ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported here emphasize the potential of bacteria in combination with suitable fermentation technology in industrial scale. The innovation compared with traditional systems is characterized by excellent long-term stability, high volumetric productivity and other technological advantages.     

Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy

Asoprisnil is a novel selective steroid receptor modulator that shows unique pharmacodynamic effects in animal models and humans. Asoprisnil, its major metabolite J912, and structurally related compounds represent a new class of progesterone receptor (PR) ligands that exhibit partial agonist and antagonist activities in vivo. Asoprisnil demonstrates a high degree of receptor and tissue selectivity, with high-binding affinity for PR, moderate affinity for glucocorticoid receptor (GR), low affinity for androgen receptor (AR), and no binding affinity for estrogen or mineralocorticoid receptors. In the rabbit endometrium, both asoprisnil and J912 induce partial agonist and antagonist effects. Asoprisnil induces mucification of the guinea pig vagina and has pronounced anti-uterotrophic effects in normal and ovariectomized guinea pigs. Unlike antiprogestins, asoprisnil shows only marginal labor-inducing activity during mid-pregnancy and is completely ineffective in inducing preterm parturition in the guinea pig. Asoprisnil exhibits only marginal antiglucocorticoid activity in transactivation in vitro assays and animal models. In male rats, asoprisnil showed weak androgenic and anti-androgenic properties. In toxicological studies in female cynomolgus monkeys, asoprisnil treatment abolished menstrual cyclicity and endometrial atrophy. Early clinical studies of asoprisnil in normal volunteers demonstrated a dose-dependent suppression of menstruation irrespective of the effects on ovulation, with no change in basal estrogen concentrations and no antiglucocorticoid effects. Unlike progestins, asoprisnil does not induce breakthrough bleeding. With favorable safety and tolerability profiles thus far, asoprisnil appears promising as a novel treatment of gynecological disorders, such as uterine fibroids and endometriosis.     

Development of a S. cerevisiae whole cell biocatalyst for in vitro sialylation of oligosaccharides

Absence of sialylation on recombinant glycoproteins compromises their efficacy as therapeutic agents, as it results in rapid clearance from the human bloodstream. To circumvent this, several strategies are followed, including the implementation of a post-secretion glycosylation step. In this paper we describe the engineering of yeast cells expressing active surface exposed Trypanosoma cruzi trans-sialidase (TS) fused to the yeast Aga2 protein, and the use of this yeast in the sialylation of synthetic oligosaccharides. In an attempt to improve overall protein accessibility on the yeast surface, we abolished hyperglycosylation on the yeast cell wall proteins. This was achieved by disrupting the OCH1 gene of the TS surface expressing strain, which resulted in increased enzymatic activity. Using a fluorescence-based activity assay and DSA-FACE structural analysis, we obtained almost complete conversion to a fully sialylated acceptor, whereas in the wild type situation this conversion was only partial. Increasing protein accessibility on the yeast surface by modifying the glycosylation content thus proved to be a valuable approach in increasing the cell wall associated activity of an immobilised enzyme, hence resulting in a more effective biocatalyst system.     

Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Disproportionate Contribution of Riparian Inputs to Organic Carbon Pools in Freshwater Systems

A lack of appropriate proxies has traditionally hampered our ability to distinguish riverine organic carbon (OC) sources at the landscape scale. However, the dissection of C₄ grasslands by C₃-enriched riparian vegetation, and the distinct carbon stable isotope signature (δ¹³C) of these two photosynthetic pathways, provides a unique setting to assess the relative contribution of riparian and more distant sources to riverine C pools. Here, we compared δ¹³C signatures of bulk sub-basin vegetation (δ¹³C_VEG) with those of riverine OC pools for a wide range of sites within two contrasting river basins in Madagascar. Although C₃-derived carbon dominated in the eastern Rianala catchment, consistent with the dominant vegetation, we found that in the C₄-dominated Betsiboka basin, riverine OC is disproportionately sourced from the C₃-enriched riparian fringe, irrespective of climatic season, even though δ¹³C_VEG estimates suggest as much as 96% of vegetation cover in some Betsiboka sub-basins may be accounted for by C₄ biomass. For example, δ¹³C values for river bed OC were on average 6.9 ± 2.7‰ depleted in ¹³C compared to paired estimates of δ¹³C_VEG. The disconnection of the wider C₄-dominated basin is considered the primary driver of the under-representation of C₄-derived C within riverine OC pools in the Betsiboka basin, although combustion of grassland biomass by fire is likely a subsidiary constraint on the quantity of terrestrial organic matter available for export to these streams and rivers. Our findings carry implications for the use of sedimentary δ¹³C signatures as proxies for past forest-grassland distribution and climate, as the C₄ component may be considerably underestimated due to its disconnection from riverine OC pools.     

Ligation of Signal Inhibitory Receptor on Leukocytes-1 Suppresses the Release of Neutrophil Extracellular Traps in Systemic Lupus Erythematosus

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of systemic Lupus erythematosus (SLE), since netting neutrophils release potentially immunogenic autoantigens including histones, LL37, human neutrophil peptide (HNP), and self-DNA. In turn, these NETs activate plasmacytoid dendritic cells resulting in aggravation of inflammation and disease. How suppression of NET formation can be targeted for treatment has not been reported yet. Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is a surface molecule exclusively expressed on phagocytes. We recently identified SIRL-1 as a negative regulator of human neutrophil function. Here, we determine whether ligation of SIRL-1 prevents the pathogenic release of NETs in SLE. Peripheral blood neutrophils from SLE patients with mild to moderate disease activity and healthy donors were freshly isolated. NET release was assessed spontaneously or after exposure to anti-neutrophil antibodies or plasma obtained from SLE patients. The formation of NETs was determined by microscopic evaluation using DNA dyes and immunostaining of NET components, as well as by live cell imaging. We show that SLE neutrophils spontaneously release NETs. NET formation is enhanced by stimulation with antibodies against LL37. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and MEK-ERK signaling prevents NET release in response to these antibodies. Signaling via the inhibitory receptor SIRL-1 was induced by ligation with anti-SIRL-1 specific antibodies. Both spontaneous and anti-neutrophil antibody-induced NET formation is suppressed by engagement of SIRL-1. Furthermore, NET release by healthy neutrophils exposed to SLE plasma is inhibited by SIRL-1 ligation. Thus, SIRL-1 engagement can dampen spontaneous and anti-neutrophil antibody-induced NET formation in SLE, likely by suppressing NAPDH oxidase and MEK-ERK activity. Together, these findings reveal a regulatory role for SIRL-1 in NET formation, potentially providing a novel therapeutic target to break the pathogenic loop in SLE.     

In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.     

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ³², was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Marker-free transgenic plants through genetically programmed auto-excision

We present here a vector system to obtain homozygous marker-free transgenic plants without the need of extra handling and within the same time frame as compared to transformation methods in which the marker is not removed. By introducing a germline-specific auto-excision vector containing a cre recombinase gene under the control of a germline-specific promoter, transgenic plants become genetically programmed to lose the marker when its presence is no longer required (i.e. after the initial selection of primary transformants). Using promoters with different germline functionality, two modules of this genetic program were developed. In the first module, the promoter, placed upstream of the cre gene, confers CRE functionality in both the male and the female germline or in the common germline (e.g. floral meristem cells). In the second module, a promoter conferring single germline-specific CRE functionality was introduced upstream of the cre gene. Promoter sequences used in this work are derived from the APETALA1 and SOLO DANCERS genes from Arabidopsis (Arabidopsis thaliana) Columbia-0 conferring common germline and single germline functionality, respectively. Introduction of the genetic program did not reduce transformation efficiency. Marker-free homozygous progeny plants were efficiently obtained, regardless of which promoter was used. In addition, simplification of complex transgene loci was observed.