The Intestinal Eukaryotic Virome in Healthy and Diarrhoeic Neonatal Piglets

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of European countries. The aim of the present study was to use viral metagenomics to examine a potential viral involvement in this diarrhoea and to describe the intestinal virome with focus on eukaryotic viruses. Samples from the distal jejunum of 50 diarrhoeic and 19 healthy piglets from 10 affected herds were analysed. The viral fraction of the samples was isolated and nucleic acids (RNA and DNA fractions) were subjected to sequence independent amplification. Samples from diarrhoeic piglets from the same herds were pooled whereas samples from healthy piglets were analysed individually. In total, 29 clinical samples, plus two negative controls and one positive control consisting of a mock metagenome were sequenced using the Ion Torrent platform. The resulting sequence data was subjected to taxonomic classification using Kraken, Diamond and HMMER. In the healthy specimens, eight different mammalian virus families were detected (Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picornaviridae, and Reoviridae) compared to four in the pooled diarrhoeic samples (Anelloviridae, Circoviridae, Picornaviridae, and Reoviridae). It was not possible to associate a particular virus family with the investigated diarrhoea. In conclusion, this study does not support the hypothesis that the investigated diarrhoea was caused by known mammalian viruses. The results do, however, indicate that known mammalian viruses were present in the intestine as early as 24–48 hours after birth, indicating immediate infection post-partum or possibly transplacental infection.     

What if fertility decline is not permanent? The need for an evolutionarily informed approach to understanding low fertility

‘Demographic transition theory’ assumes that fertility decline is irreversible. This commonly held assumption is based on observations of recent and historical reductions in fertility that accompany modernization and declining mortality. The irreversibility assumption, however, is highly suspect from an evolutionary point of view, because demographic traits are at least partially influenced by genetics and are responsive to social and ecological conditions. Nonetheless, an inevitable shift from high mortality and fertility to low mortality and fertility is used as a guiding framework for projecting human population sizes into the future. This paper reviews some theoretical and empirical evidence suggesting that the assumption of irreversibility is ill-founded, at least without considerable development in theory that incorporates evolutionary and ecological processes. We offer general propositions for how fertility could increase in the future, including natural selection on high fertility variants, the difficulty of maintaining universal norms and preferences in a large, diverse and economically differentiated population, and the escalating resource demands of modernization.     

Marine bioprocess engineering: the missing link to commercialization

Success of US biotechnology has been and continues to be dependent on new discoveries and their timely transformation into useful products through bioprocess engineering and a systems approach. Bioprocess engineering is an essential element of ‘generic applied’ or ‘precompetitive’ research. For marine biotechnology, like biopharmaceutical biotechnology, bioprocess engineering represents the key. The many hundreds of tantalizing bioactive compounds discovered and isolated from varied marine organisms over the past decades have led to only minimal commercialization due to the limited availability of the compounds in question. To address international competitiveness and the revitalization of key US industries, the National Science Foundation launched the Engineering Research Centers Program in the mid 1980s. The essential feature of this program is a partnership among academia, industry and the government to develop next-generation technology through cutting-edge research, relevant education and innovative technology transfer. MarBEC (Marine Bioproducts Engineering Center) is a recently established multi-disciplinary engineering-science cooperative effort of the University of Hawaii and the University of California at Berkeley. Additional partners include three federal laboratories—Argonne National Laboratory, the Edgewood Research, Development and Engineering Center and the Eastern Regional Research Center of the US Department of Agriculture—and the Bishop Museum. MarBEC's research program consists of four major thrusts: Production Systems; Marine Bioproducts and Bioresources; Separation and Conversion; and Bioproduct Formulation.     

Zur symbiose zwischen actinien und paguriden

Ohne Zusammenfassung     

A process‐based model supports an association between dispersal and the prevalence of species traits in tropical reef fish assemblages

Habitat dynamics interacting with species dispersal abilities could generate gradients in species diversity and prevalence of species traits when the latter are associated with species dispersal potential. Using a process‐based model of diversification constrained by a dispersal parameter, we simulated the interplay between reef habitat dynamics during the past 140 million years and dispersal, shaping lineage diversification history and assemblage composition globally. The emerging patterns from the simulations were compared to current prevalence of species traits related to dispersal for 6315 tropical reef fish species. We found a significant spatial congruence between the prevalence of simulated low dispersal values and areas with a large proportion of species characterized by small adult body size, narrow home range mobility behaviour, pelagic larval duration shorter than 21 days and diurnal activity. Species characterized by such traits were found predominantly in the Indo‐Australian Archipelago and the Caribbean Sea. Furthermore, the frequency distribution of the dispersal parameter was found to match empirical distributions for body size, PLD and home range mobility behaviour. Also, the dispersal parameter in the simulations was associated to diversification rates and resulted in trait frequency matching empirical distributions. Overall, our findings suggest that past habitat dynamics, in conjunction with dispersal processes, influenced diversification in tropical reef fishes, which may explain the present‐day geography of species traits.     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

The Two Plastidial Starch-Related Dikinases Sequentially Phosphorylate Glucosyl Residues at the Surface of Both the A- and B-Type Allomorphs of Crystallized Maltodextrins But the Mode of Action Differs

In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of monophosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases.In terms of quantity, starch is one of the most prominent photosynthesis-derived products. The global starch production by land plants has been estimated to be approximately 2,850 million tons per year (Burrell, 2003). Starch is highly relevant for nutrition in animals and humans, but it is also used for many industrial applications, such as additives in paper or textiles and in pharmacy products as well. In addition, starch appears to be increasingly important as a photosynthesis-based renewable energy source that can be converted into technologically relevant products such as bioethanol and hydrogen (Hannah and James, 2008; Zhang et al., 2008).Native starch is formed as a water-insoluble particle called a granule that is thought to comprise two types of polyglucans, amylopectin and amylose. The latter is an almost unbranched α-1,4-glucan and usually is the minor constituent of the starch particle, accounting for 10% to 35% of the total starch dry weight (Ball, 2000). However, in some mutants, the relative amylose content is strongly diminished, resulting in an essentially amylose-free starch (such as in the waxy mutant of maize [Zea mays]), or, alternatively, it is increased, forming up to 70% of the starch mass (e.g. in the amylose extender mutant from maize; Gérard et al., 2001). Nevertheless, in wild-type starches, amylopectin typically is the major constituent that also is essential for the molecular organization of the glucans within the entire starch granule (Ball and Morell, 2003). Like glycogen, amylopectin is a branched α-glucan with 4% to 6% of the inter-Glc linkages being α-1,6-bonds (Ball, 2000); however, as opposed to glycogen, the branching points occur as intramolecular clusters. Due to the length distribution of the side chains and the clustering of the branching points, neighboring glucan chains are capable of forming highly ordered double helices (Smith, 2001; Zeeman et al., 2002).As revealed by x-ray diffraction analysis, two major native starch structures are known that differ in the arrangement of the double helices. The A-type allomorph, which is typical of wild-type cereal starches but also occurs in lower plants, is more compact, as compared with the B type, and consists of flat layers of double helices. By contrast, in the B-type allomorph, six double helices are thought to surround a central cavity that is filled with water molecules. The B-type allomorph is found in starch synthesized by dicotyledonal storage organs, such as potato (Solanum tuberosum) tubers, in some high-amylose starches from cereal mutants (Gallant et al., 1997; Gérard et al., 2001), and in assimilatory starches from potato and Arabidopsis (Arabidopsis thaliana) as well (Hejazi et al., 2008). Legume starches are believed to represent another allomorph that is designated the C type. However, this allomorph is actually a mixture of both the A- and B-type crystallites within a single native starch particle rather than a third distinct type of the double helical arrangement (Imberty et al., 1991; Bogracheva et al., 2001).It should be noted that both the A- and B-type allomorphs of native starch granules often contain, as a minor constituent, an additional crystal structure designated the V type. Unlike the A- and B-type allomorphs, the V type is assumed to arise from single amylose helices, some of which are complexed with endogenous granular lipids. When estimated for the dry state, the V-type crystal structure accounts for only a small percentage of the total starch granule crystallinity (Lopez-Rubio et al., 2008).The physical structure of the native starch particle is likely to have important biochemical implications, as it affects the performance of carbohydrate-active enzymes and, thereby, the transition of carbohydrates from the solid phase to the soluble phase. This conclusion has been reached by in vitro experiments demonstrating that the pancreas α-amylase hydrolyzes A-type starch faster than the B-type counterpart (Gérard et al., 2001).Another metabolically important feature of amylopectin is the occurrence of covalent modification by phosphate esters that are found in a small proportion of the glucosyl residues. Most frequently phosphorylation occurs at the C6 position of the glucosyl residue, but C3 and, to a minor extent, C2 can also be esterified (Hizukuri et al., 1970). Recently, evidence has been presented that the esterification of the C6 and C3 positions of glucosyl residues differs in the structural effects on the neighboring inter-Glc bonds (Hansen et al., 2009). Phosphorylation at C6 is mediated by the recently identified α-glucan, water dikinase (GWD; EC 2.7.9.4), which utilizes ATP as dual phosphate donor and three distinct acceptors, two of which are sequentially used. The enzyme transfers the terminal phosphate group to water (thereby forming orthophosphate) and the β-phosphate group first to a conserved His residue within the catalytic domain of the monomeric GWD and, subsequently, to the C6 target of the glucosyl residue to be phosphorylated (Ritte et al., 2002, 2006). Phosphorylation at C3 is catalyzed by a second dikinase, designated phosphoglucan, water dikinase (PWD; EC 2.7.9.5; Ritte et al., 2006). The amino acid sequence of the catalytic (C-terminal) domain of PWD shares similarity with that of GWD, and in principle, the PWD-mediated catalysis follows the same mode of action as GWD, including the transient autophosphorylation at a conserved His residue (Baunsgaard et al., 2005; Kötting et al., 2005). However, PWD deviates from GWD in the amino acid sequence of the N-terminal domain, especially in the carbohydrate-binding region. PWD possesses a single carbohydrate-binding module that has been grouped into the family CBM20 (Machovič and Janaček, 2006a, 2006b). By contrast, the N-terminal domain of GWD contains two putative carbohydrate-binding motifs similar to those of an α-amylase that presumably is located in the chloroplasts (Yu et al., 2005). However, the structure of these motifs is still not known; therefore, a sequence-based prediction of the actual carbohydrate target is not yet possible.GWD- and PWD-deficient Arabidopsis mutants possess to some extent similar but not equal phenotypes. Leaves of GWD-deficient lines (which contain essentially unchanged levels of functional PWD) have starch levels that are at least five times higher than those of the wild type and remain high even after prolonged darkness. Growth of the entire plant is strongly compromised. The phenotype of PWD-deficient mutants (which express functional GWD) is less severe, as growth is only slightly diminished and transitory starch levels are elevated but not as strongly as in the GWD-deficient lines. Mutants lacking functional PWD can degrade transitory starch, but net degradation occurs at a lower rate as compared with wild-type plants (Kötting et al., 2005). These data clearly indicate that, in vivo, PWD cannot substitute for GWD and that glucosyl 6-phosphate residues are involved in a more strict control of the starch turnover as compared with the C3 phosphate esters.When considering the metabolic function(s) of starch phosphorylation, it should be noted that phosphorylation occurs during both net starch synthesis and degradation, although the rates of phosphorylation are likely to be different (Nielsen et al., 1994; Ritte et al., 2004). It is reasonable, therefore, to assume that starch phosphorylation exerts an important role in the entire transitory starch metabolism, rather than being functional only during the degrading process (and, consequently, the starch-related dikinases cannot, in a strict sense, be considered as “starch-degrading enzymes”).Depending on the botanical source, the degree of starch phosphorylation varies strongly. In potato tuber starch, approximately 0.1% to 0.5% of the glucosyl residues are phosphorylated (Ritte et al., 2002), and this value is considered to be indicative of a high level of phosphorylation. By contrast, cereal starches contain a far lower relative phosphate content that often is close to the limit of detection (approximately 0.002%; Glaring et al., 2006). In principle, these differences could be due to different rates of phosphorylation, as catalyzed by the two starch-related dikinases, and this assumption seems to be supported by the observation that, in general, starches of the B-type allomorph appear to have a higher degree of phosphorylation as compared with those of the A-type allomorph. If so, the dikinases may preferentially act on the B-type allomorph. Alternatively, the phosphorylation catalyzed by the two dikinases could be balanced by counteracting phosphatases, such as SEX4. This plastidial enzyme has been shown to act as a (phospho)glucan phosphatase that is involved in leaf starch metabolism (Kötting et al., 2009). If antagonistic enzyme activities are taken into consideration, the actual level of starch phosphorylation is determined by the rate of both phosphorylation and the subsequent hydrolysis of phosphate esters and, consequently, does not necessarily reflect the action of the starch-related dikinases.Recently, crystallized maltodextrins (MD_cryst) have been prepared that, by using x-ray diffraction, were identified as being the B-type allomorph and to possess a highly ordered structure (which exceeds that of native starch granules). MD_cryst have been applied as a substrate for a recombinant GWD from potato. Using a carefully optimized assay, the rate of phosphorylation was by far higher than that observed with any other carbohydrate substrate, such as native starch granules or starch-derived polysaccharides. By contrast, solubilization by heat treatment of the MD_cryst almost completely abolished the activity of GWD. Phosphorylation resulted in the formation of singly, doubly, and triply phosphorylated glucans and favored the solubilization of both neutral glucans and phosphoglucans (Hejazi et al., 2008). Recombinant PWD also phosphorylated MD_cryst, provided the MD_cryst had been prephosphorylated by GWD and were not solubilized by heat treatment (Hejazi et al., 2008).Because of the high phosphorylation rates and the phosphorylation pattern obtained, MD_cryst are a suitable model carbohydrate that mimics phosphorylation-relevant features of highly ordered regions within the native starch granule. It allows study of the action of the two starch-related dikinases and the transition of carbohydrates from the solid to the soluble state without any other starch-related enzyme being required.Until now, only the B-type allomorph of the MD_cryst has been applied as substrate of the two dikinases. Using native starch granules as a target, the rates of phosphorylation as obtained with recombinant GWD varied largely within the B-type allomorph (Hejazi et al., 2008); therefore, it is reasonable to assume that additional but largely unknown features of the native starch granule also strongly affect the action of GWD. This implies that any preference or specificity of the starch-related dikinases for a given allomorph can be analyzed most convincingly if MD_cryst preparations representing both the B- and A-type allomorphs are available.In this study, we used two MD_cryst preparations that are indistinguishable in their oligoglucan patterns but differ in the physical arrangement of the double helices and represent the highly ordered A- and B-type allomorphs. Using these two MD_cryst preparations, we analyzed the action of the two starch-related dikinases. The size distribution of the MD_cryst particles has been determined using the Coulter counter, and surface properties of both allomorphs were monitored by scanning electron microscopy. Thermal stability of the two allomorphs was analyzed by measuring the temperature dependence of light scattering. Finally, the phosphorylation-dependent solubilization of both allomorphs and the transition of (phospho)glucans into the soluble state have been studied.