Maintaining high anaerobic succinic acid productivity by product removal

During dual-phase fermentations using Escherichia coli engineered for succinic acid production, the productivity and viable cell concentration decrease as the concentration of succinic acid increases. The effects of succinic acid on the fermentation kinetics, yield, and cell viability were investigated by resuspending cells in fresh media after selected fermentation times. The cellular succinic acid productivity could be restored, but cell viability continuously decreased throughout the fermentations by up to 80% and subsequently the volumetric productivity was reduced. Omitting complex nutrients in the resuspension media had no significant effect on cellular succinate productivity and yield, although the viable cell concentration and thus the volumetric productivity was reduced by approximately 20%. By resuspending the cells, the amount of succinate produced during a 100-h fermentation was increased by more than 60%. The results demonstrate that by product removal succinic acid productivity can be maintained at high levels for extended periods of time.     

2‐D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum ΔTri5 infection and grain development

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase‐inhibiting proteins (XIPs), and thaumatin‐like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a ΔTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2‐D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum ΔTri5. Some (iso)forms were up‐regulated, whereas others were down‐regulated. This pathogen‐specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI‐ and XIP‐type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non‐glycosylated XIP forms increased more strongly than their glycosylated counterparts.     

Modeling Lignin Polymerization. I. Simulation Model of Dehydrogenation Polymers

Lignin is a heteropolymer that is thought to form in the cell wall by combinatorial radical coupling of monolignols. Here, we present a simulation model of in vitro lignin polymerization, based on the combinatorial coupling theory, which allows us to predict the reaction conditions controlling the primary structure of lignin polymers. Our model predicts two controlling factors for the β-O-4 content of syringyl-guaiacyl lignins: the supply rate of monolignols and the relative amount of supplied sinapyl alcohol monomers. We have analyzed the in silico degradability of the resulting lignin polymers by cutting the resulting lignin polymers at β-O-4 bonds. These are cleaved in analytical methods used to study lignin composition, namely thioacidolysis and derivatization followed by reductive cleavage, under pulping conditions, and in some lignocellulosic biomass pretreatments.Lignins are aromatic polymers that are predominantly present in secondarily thickened cell walls. These polymers make the cell wall rigid and impervious, allowing transport of water and nutrients through the vascular system and protecting plants against microbial invasion. Lignins are heterogeneous polymers derived from phenylpropanoid monomers, mainly the hydroxycinnamyl alcohols coniferyl alcohol (G-monomer) and sinapyl alcohol (S-monomer) and minor amounts of p-coumaryl alcohol (H-monomer). These monolignols differ in their degree of aromatic methoxylation (-OCH₃ group; Fig. 1). The resulting units in the lignin polymer are the guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units. They are linked by a variety of chemical bonds (Fig. 2) that have different chemical properties (Boerjan et al., 2003; Ralph et al., 2004; Vanholme et al., 2008).Open in a separate windowFigure 1.Chemical structures of three monolignols. A, H-monomer (p-coumaryl alcohol). B, G-monomer (coniferyl alcohol). C, S-monomer (sinapyl alcohol). G- and S-monomers are considered in our simulations. The G-monomer is methoxylated (-OCH₃ group) on position 3, and the S-monomer is methoxylated on positions 3 and 5.Open in a separate windowFigure 2.Chemical structures resulting from the possible bonding between two monomers (A) or a monomer and the bindable end of an oligomer (B). X and Y in the monomers denote the absence (for a G-unit) or presence (for an S-unit) of a methoxyl group at position 5 (see Fig. 1). The red line indicates the bonds generated by couplings of the B position and B, 4, or 5 position.Lignification is the process by which monomers and/or oligomers are polymerized via radical coupling reactions and typically occurs after the polysaccharides have been laid down in the cell wall. Lignin composition varies among cell types and can even be different in individual cell wall layers (Ruel et al., 2009). Lignin composition is also influenced by environmental conditions; for example, lignin in compression wood is enriched in H-units (Timell, 1986). Hence, both developmental and environmental parameters influence the composition and thus the structure of the lignin polymer (Boerjan et al., 2003; Ralph et al., 2004).Lignin is one of the main negative factors in the conversion of lignocellulosic plant biomass into pulp and bioethanol (Lynd et al., 1991; Hill et al., 2006). In these processes, lignin needs to be degraded by chemical or mechanical processes that are expensive and often environmentally polluting. Hence, major research efforts are devoted toward understanding lignin biosynthesis and structure. It has already been shown that reducing lignin content and modifying its composition in transgenic plants can result in dramatic improvements in pulping efficiency (Pilate et al., 2002; Baucher et al., 2003; Huntley et al., 2003; Leplé et al., 2007) and in the conversion of biomass into bioethanol (Stewart et al., 2006; Chen and Dixon, 2007; Custers, 2009). These altered biomass properties are related to the alterations in lignin composition and structure in terms of the frequencies of the lignin units and the bond types connecting them and possibly also their interaction with hemicelluloses (Ralph et al., 2004; Ralph, 2006).To study the parameters that influence lignin structure, lignin polymerization has been mimicked in vitro by experiments with dehydrogenation polymers (DHPs; Terashima et al., 1995). Indeed, lignification can be mimicked by oxidizing monolignols using a peroxidase, such as horseradish peroxidase (HRP), and supplying its cofactor hydrogen peroxide, producing synthetic DHP lignins. Monolignol oxidation can also be achieved without enzymes (e.g. by using transition metal one-electron oxidants, such as copper acetate). Some of these biomimetic DHPs have been suggested to be better models for wood lignins than HRP-generated DHPs (Landucci, 2000).In DHP experiments, the monolignols are either added in bulk (Zulauf experiment) or dropwise (Zutropf experiment) to the reaction mixture, yielding lignin polymers with very different bond frequencies (Freudenberg, 1956). Zutropf experiments approach the in vivo formation of lignin, which depends on the slow introduction of monolignols into the wall matrix via diffusion to the site of incorporation (Hatfield and Vermerris, 2001). Because the exact reaction conditions are known, such in vitro experiments have provided insight into the lignification process in planta. In this way, numerous factors were shown to influence lignin structure, including the relative supply of the monolignols, the pH, the presence of polysaccharides, hydrogen peroxide concentrations, and cell wall matrix elements in general (Grabber et al., 2003; Vanholme et al., 2008).Computer simulations of lignin polymerization can help explain and predict lignin structure from low-level chemical kinetic factors, including subunit-coupling probabilities and monolignol synthesis rates. Such models are helpful in explaining the mechanism behind a range of controlling factors identified in the experimental work, including (1) the ratio of coniferyl versus sinapyl alcohol monolignols, (2) the monolignol supply rate, and (3) the abundance of alternative monomers present during lignin biosynthesis in mutants and transgenics. Thus, computer models will also help in suggesting new targets for controlled lignin biosynthesis.Here, we propose a simulation model of synthetic lignin polymerization that is based upon an emerging consensus from a variety of observations and derives from a series of previous models of lignin polymerization (Glasser and Glasser, 1974; Glasser et al., 1976; Jurasek, 1995; Roussel and Lim, 1995). Our model uses a symbolic grammar to describe a constructive dynamical system (Fontana, 1992) or a rule-based system (Feret et al., 2009) in which it is not necessary to define all possible products in advance. We assume that G- and S-monomers and newly formed oligomers couple in a well-mixed medium, depending on coupling rules and experimentally measured coupling probabilities. To develop the model, we have used information from DHP experiments rather than natural lignins, as they are formed in a well-mixed medium and their reaction conditions are well known (e.g. the influx rate of monomers). Using information from natural lignin would have further complicated our model, as the structures of natural lignin polymers are influenced by many factors, including the possible involvement of dirigent proteins (Davin and Lewis, 2005), steric hindrance by polysaccharides, spatiotemporal regulation, and modifications during isolation procedures (Boerjan et al., 2003; Ralph et al., 2004).Using our simulation models, we study how putative controlling factors of lignin primary structure, including the influx rate of monomers and the relative amount of S-monomers, affect in silico lignin synthesis, and we compare our predictions with in vitro experiments. To predict the degradability of lignins formed in our simulations, we apply an in silico thioacidolysis, which cleaves the polymers at their β-O-4 positions. This simulates the molecular action of two of the most used methods to analyze lignin composition, thioacidolysis (Lapierre, 1993; Baucher et al., 2003) and derivatization followed by reductive cleavage (Lu and Ralph, 1997). The G+S-monomer yield is often taken as a reflection of the fraction of units bound by β-O-4 bonds. Cleavage of β-O-4 bonds is also the most important reaction in kraft pulping of wood (Baucher et al., 2003). The model predicts from first principles (1) that DHP lignins formed under Zutropf conditions have a higher β-O-4 content than those formed under Zulauf conditions, (2) that DHP lignins formed with high S content have a higher β-O-4 content than those formed with high G content, and (3) that a higher β-O-4 content does not necessarily reduce the average length of lignin fragments generated during in silico thioacidolysis.     

The MAGUK protein MPP7 binds to the polarity protein hDlg1 and facilitates epithelial tight junction formation

Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.     

Frequency of Bt resistance alleles in Helicoverpa armigera in the Xinjiang cotton-planting region of China

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a key insect pest of cotton in Xinjiang cotton-planting region of northwest China. In this region, cotton is grown on average ≈ 1.65 million ha (1.53 ≈ 1.80 million ha) annually in largely monoculture agricultural landscapes, similarly to cropping systems in the United States or Australia. Under such cropping regimes, naturally occurring refuges (with non-Bt crops) may be insufficient to prevent H. armigera resistance development to Bt toxins. Therefore, we assessed frequency of alleles conferring resistance to Cry1Ac toxin of F(1) and F(2) offspring of H. armigera isofemale lines from two distinct localities in the region during 2005-2009. More specifically, a total of 224 isofemale lines was collected from Korla County (≈ 70% Bt cotton adoption) and 402 lines from Shache County (≈ 5% Bt cotton planting). Subsequent offspring was screened on Cry1Ac artificial diet. From 2005 to 2009, resistance gene frequency in Korla fluctuated between 0.0000 and 0.0040, while being 0.0000-0.0008 in individuals collected from Shache, and there were no significant increases in both counties from 2005 to 2009. Relative average development rates (RADRs) of larvae in F(1) tests showed significant increases from Korla, but not in Shache. RADR of F(1) larvae is significantly correlated with RADR of F(2) offspring, indicating genetic variation in response to toxin in field H. armigera population. Although the occurrence of Cry1Ac resistance alleles was low in Xinjiang cotton-planting region of China, particular attention should be given to H. armigera resistance development in Korla County.     

Dental caries in wild primates: Interproximal cavities on anterior teeth

Dental caries has been reported in a variety of primates, although it is still considered rare in wild populations. In this study, 11 catarrhine primate taxa (n = 339 individuals; 7946 teeth) were studied for the presence of caries. A differential diagnosis of lesions in interproximal regions of anterior teeth was undertaken, since they had been previously described as both carious and non-carious in origin. Each permanent tooth was examined macroscopically, with severity and position of lesions recorded. Two specimens were examined further, using micro-CT scans to assess demineralization. Differential diagnosis confirmed the cariogenic nature of interproximal cavities on anterior teeth (ICATs). Overall results show 3.3% of all teeth (i.e., anterior and posterior teeth combined) were carious (n = 262), with prevalence varying among species from 0% to >7% of teeth affected. Those with the highest prevalence of ICATs include Pan troglodytes verus (9.8% of anterior teeth), Gorilla gorilla gorilla (2.6%), Cercopithecus denti (22.4%), Presbytis femoralis (19.5%), and Cercopithecus mitis (18.3%). ICATs make up 87.9% of carious lesions on anterior teeth. These results likely reflect dietary and food processing differences among species, but also between the sexes (e.g., 9.3% of all female P. troglodytes verus teeth were carious vs. 1.8% in males). Processing cariogenic fruits and seeds with the anterior dentition (e.g., wadging) likely contributes to ICAT formation. Further research is needed in living primate populations to ascertain behavioral/dietary influences on caries occurrence. Given the presence of ICATs in frugivorous primates, their diagnosis in archaeological and paleontological specimens may shed light on diet and food processing behaviors in fossil primates.     

Prokaryotic communities adapted to microhabitats on the Indian lotus (Nelumbo nucifera) growing in the high-altitude urban Dal Lake

International Microbiology - Indian lotus (Nelumbo nucifera) is one of the dominant aquatic plants cultivated in Dal Lake, situated at 1586 m above mean sea level (MSL) in the northeast of...     

The palaeoecology of the Upper Ndolanya Beds at Laetoli,Tanzania

The palaeoecology of the fauna from the Ndolanya Beds, Laetoli, Tanzania, has been analysed to reconstruct the environment of this 2.6 Ma site. Community profiles have been constructed in relation to three variables that carry ecological meaning: body weight, locomotor adaptations and feeding preferences. Comparing the similarities and differences in the structure of the fossil faunas with those of modern environments allows us to draw inferences about palaeoenvironmental conditions, and this is based on a comparative dataset of 15 modern environments (44 localities) covering a wide range of climatic and ecological conditions across Asia, Africa and Central America. In addition, 16 fossil sites in East and South Africa have been analysed in the same way, and both sets of comparative data have been used as the basis for comparison with an ecological diversity analysis of the fauna from the Upper Ndolanya Beds. The Ndolanya fauna is characterized by a predominance of medium to large sized terrestrial and herbivorous species.There is evidence of taphonomic bias that eliminated many of the smaller species. A comparison of multivariate analyses of 23 selected modern localities conducted both with and without the small species included, indicates that the loss of these species does not adversely influence the results of an ecological diversity analysis. The evidence suggests that at the time of the deposition of the Ndolanya Beds the Laetoli region was a semi-arid bushland. This is considerably drier and more open than is suggested for the earlier Laetolil Beds.