Preparative purification of polyethylene glycol derivatives with polystyrene-divinylbenzene beads as chromatographic packing

A clear and powerful chromatographic approach to purify polyethylene glycol derivatives at a preparative scale was reported, which was based on the polystyrene-divinylbenzene beads with ethanol/water as eluants. The validity of this method was verified with the reaction mixture of mPEG-Glu and mPEG propionaldehyde diethylacetal (ALD-PEG) as the model. The target products were one-step achieved with the purity of >99% on the polymer resins column at gram scale. The method developed was free from such disadvantages as utility of toxic solvent and narrow application scope, which was combined with conventional approaches. The method developed provided an appealing and attractive alternative methods for purification of PEG derivatives at a preparative scale.     

Comparison of covalent immobilization of amylase on polystyrene pellets with pentaethylenehexamine and pentaethylene glycol spacers

α-Amylase from Aspergillus oryzae was covalently immobilized onto polystyrene pellets with pentaethylenehexamine (PS-PEHA-Ald) and pentaethylene glycol (PS-PG-Ald) carrying a terminal aldehyde group. Optimum immobilization occured at pH 8.0 and 25 °C, and at pH 7.0 and 35 °C for PS-PEHA-Ald and PS-PG-Ald, respectively. PS-PEHA-Ald immobilized enzyme retained approximately 75% of the initial activity over 45 days of storage, 70% of the initial activity after nine runs of recycling and displayed the better resistance to detrimental metal ions. PS-PG-Ald immobilized enzyme retained approximately 50% of the initial activity in 8h at 70 °C. The catalytic efficiencies of PS-PEHA-Ald immobilized and PS-PG-Ald immobilized amylase were 1.42 and 1.29 times higher than that of native enzyme. The activation energy of the reaction mediated by the amylase was reduced by 58.1% and 57.3% when PS-PEHA-Ald and PS-PG-Ald used as support respectively.     

Biofilm stratification during simultaneous nitrification and denitrification (SND) at a biocathode

The aeration of the cathode compartment of bioelectrochemical systems (BESs) was recently shown to promote simultaneous nitrification and denitrification (SND). This study investigates the cathodic metabolism under different operating conditions as well as the structural organization of the cathodic biofilm during SND. Results show that a maximal nitrogen removal efficiency of 86.9 ± 0.5%, and a removal rate of 3.39 ± 0.08 mg N L⁻¹ h⁻¹ could be achieved at a dissolved oxygen (DO) level of 5.73 ± 0.03 mg L⁻¹ in the catholyte. The DO levels used in this study are higher than the thresholds previously reported as detrimental for denitrification. Analysis of the cathodic half-cell potential during batch tests suggested the existence of an oxygen gradient within the biofilm while performing SND. FISH analysis corroborated this finding revealing that the structure of the biofilm included an outer layer occupied by putative nitrifying organisms, and an inner layer where putative denitrifying organisms were most dominant. To our best knowledge this is the first time that nitrifying and denitrifying microorganisms are simultaneously observed in a cathodic biofilm.     

Patch configuration affects alpine plant distribution

The relative importance of niche requirements and dispersal limitation in controlling the landscape‐scale distribution of plants is still contentious. Local occurrence and abundance of alpine plants are commonly thought to be driven by abiotic site conditions due to pronounced environmental gradients over short distances. However, explicit tests of the additional role of dispersal‐related processes for alpine plant distribution patterns are lacking. Here, we combine niche‐based species distribution models with variables describing patch size and connectivity to evaluate if, besides abiotic limitations, spatial habitat configuration affects the occurrence and abundance of six plant species inhabiting patchy snowbed mosaics of the northeastern Calcareous Alps in Austria. Moreover, we assess if eventual effects of spatial patch configuration are more clearly detectable when calculating connectivity based on parameterized mechanistic dispersal kernels for both wind and animal vectors instead of using nearest neighbour metrics. We show that patch size and connectivity are significantly correlated to the occurrence of all and to the abundance of four out of six study species, although the relative importance of these variables, as compared to niche constraints, varies among species. In addition, connectivity measures derived from parameterized dispersal kernels were more closely related to occupancy, and in particular to abundance patterns than a simple nearest neighbour metric. The fitted kernels also suggest that dispersal by alpine chamois plays an important role for inter‐patch seed exchange. We conclude that, despite evident abiotic limitations, recurrent local extinctions and delayed re‐colonizations indeed play a role for the distribution of our study species, and that alpine plants may hence be less in equilibrium with their abiotic environment than commonly thought. Moreover, the relatively high long‐distance dispersal probabilities of animal kernels indicate that the ability of alpine plants to adapt their ranges to a rapidly warming climate may, among other factors, depend on the availability of dispersal services by large mammals.     

Isolation of a heavy metal-resistant 4-Chloronitrobenzene degrader Cupriavidus sp. D4 and cloning of its cnb genes

Strain D4 was isolated from the sludge of the wastewater treating system of a 4-Chloronitrobenzene (4-CNB) manufacturer. It was able to utilize 4-CNB as the sole carbon and nitrogen source for growth. Strain D4 was preliminarily identified as Cupriavidus sp. based on its physiological & biochemical characteristics and 16S rRNA gene sequence analysis. It could completely degrade 300 mg L⁻¹ of 4-CNB within 25 h under the condition of 30 °C and pH 7.0. Strain D4 could also degrade 4-CNB in presence of heavy metals including Co²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Mn²⁺and so on, therefore it was an excellent candidate for the bio-treatment of 4-CNB and heavy metals co-contaminated environments. The main 4-CNB degrading related genes (cnb A, B, Cab, D, G, Z) and arsenate resistance gene fragment of strain D4 were cloned, sequenced and analyzed, which showed high similarity with the corresponding genes of a reported 4-CNB-degrader, strain CNB-1. The cnb genes of strain D4 were located on two plasmids. This is the first report on the degradation of 4-CNB by the strain from the genus of Cupriavidus sp.     

Oleic acid decreases the expression of a cholesterol transport-related protein (NPC1L1) by the induction of endoplasmic reticulum stress in CaCo-2 cells

The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.25–1.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which may play an important role in reducing intestinal cholesterol absorption.     

<Emphasis Type="Italic">In silico</Emphasis> improvement of heterologous biosynthesis of erythromycin precursor 6-deoxyerythronolide B in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The heterologous biosynthesis of 6-deoxyerythronolide B (6dEB), a key intermediate in the biosynthesis of erythromycin, has recently been achieved in Escherichia coli, but the experimental product yield remains low. In this study, in silico strategies were adopted to evaluate and improve the biosynthesis of 6dEB in this strain. The theoretical capability of E. coli to produce 6dEB was first evaluated by analyzing the maximum theoretical molar yield (MTMY) of 6dEB utilizing three carbon sources, glucose, propionate and glycerol. Although propionate is presently most often used experimentally, our results indicated that glucose would be the most feasible substrate for 6dEB production from economic and long-term standpoints. Compared with Saccharomyces cerevisiae and Bacillus subtilis, E. coli was found to be a better heterologous host for the biosynthesis of 6dEB due to the higher MTMY value under the same conditions. Two strategies, including a flux distribution comparison analysis (FDCA) and linear minimization of metabolic adjustment based (LMOMA-based) methods, were proposed and employed for in silico strain improvement of 6dEB production, which yielded several potential gene targets for future experimental validation. In a further analysis, increasing the specific growth rate (SGR) or the non-growth associated maintenance (NGAM) was found to decrease the MTMY; while increasing the specific oxygen uptake rate (SOUR) or the specific carbon source uptake rate (SCUR) increased the MTMY. Taken together, our findings identified key factors directly affecting the MTMY of 6dEB production, which will guide future experimental research or even the industrial production of 6dEB.