A novel enzymatic microreactor with Aspergillus oryzae β‐galactosidase immobilized on silicon dioxide nanosprings

The use of silicon dioxide (SiO₂) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm² × 60 μm thick) was functionalized with γ‐aminopropyltriethoxysilane, then treated with N‐succinimidyl‐3‐(2‐pyridyldithio)‐propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (? SH) groups. SPDP‐modified β‐galactosidase from Aspergillus oryzae was immobilized on the thiolated nanosprings by reversible disulfide linkages. The enzyme‐coated nanospring mat was placed into a 175‐μm high microchannel, with the mat partially occluding the channel. The kinetics and steady‐state conversion of hydrolysis of o‐nitrophenyl β‐D ‐galactosylpyranoside at various substrate flow rates and concentrations were measured. Substantial flow was observed through the nanosprings, for which the Darcy permeability κ ≈ 3 × 10^?6 cm². A simple, one‐parameter numerical model coupling Navier‐Stokes and Darcy flow with a pseudo‐first‐order reaction was used to fit the experimental data. Simulated reactor performance was sensitive to changes in κ and the height of the nanospring mat. Permeabilities lower than 10^?8 cm² practically eliminated convective flow through the nanosprings, and substantially decreased conversion. Increasing the height of the mat increased conversion in simulations, but requires more enzymes and could cause sealing issues if grown above channel walls. Preliminary results indicate that in situ regeneration by reduction with DTT and incubation with SPDP‐modified β‐galactosidase is possible. Nanosprings provide high solvent‐accessible surface area with good permeability and mechanical stability, can be patterned into existing microdevices, and are amenable to immobilization of biomolecules. Nanosprings offer a novel and useful support for enzymatic microreactors, biosensors, and lab‐on‐chip devices. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

From Early Domesticated Rice of the Middle Yangtze Basin to Millet,Rice and Wheat Agriculture: Archaeobotanical Macro-Remains from Baligang,Nanyang Basin,Central China (6700–500 BC)

Baligang is a Neolithic site on a northern tributary of the middle Yangtze and provides a long archaeobotanical sequence from the Seventh Millennium BC upto the First Millennium BC. It provides evidence for developments in rice and millet agriculture influenced by shifting cultural affiliation with the north (Yangshao and Longshan) and south (Qujialing and Shijiahe) between 4300 and 1800 BC. This paper reports on plant macro-remains (seeds), from systematic flotation of 123 samples (1700 litres), producing more than 10,000 identifiable remains. The earliest Pre-Yangshao occupation of the sites provide evidence for cultivation of rice (Oryza sativa) between 6300–6700 BC. This rice appears already domesticated in on the basis of a dominance of non-shattering spikelet bases. However, in terms of grain size changes has not yet finished, as grains are still thinner than more recent domesaticated rice and are closer in grain shape to wild rices. This early rice was cultivated alongside collection of wild staple foods, especially acorns (Quercus/Lithicarpus sensu lato). In later periods the sites has evidence for mixed farming of both rice and millets (Setaria italica and Panicum miliaceum). Soybean appears on the site in the Shijiahe period (ca.2500 BC) and wheat (Triticum cf. aestivum) in the Late Longshan levels (2200–1800 BC). Weed flora suggests an intensification of rice agriculture over time with increasing evidence of wetland weeds. We interpret these data as indicating early opportunistic cultivation of alluvial floodplains and some rainfed rice, developing into more systematic and probably irrigated cultivation starting in the Yangshao period, which intensified in the Qujialing and Shijiahe period, before a shift back to an emphasis on millets with the Late Longshan cultural influence from the north.     

Multiple defender effects: synergistic coral defense by mutualist crustaceans

The majority of our understanding of mutualisms comes from studies of pairwise interactions. However, many hosts support mutualist guilds, and interactions among mutualists make the prediction of aggregate effects difficult. Here, we apply a factorial experiment to interactions of 'guard' crustaceans that defend their coral host from seastar predators. Predation was reduced by the presence of mutualists (15% reduction in predation frequency and 45% in volume of coral consumed). The frequency of attacks with both mutualists was lower than with a single species, but it did not differ significantly from the expected frequency of independent effects. In contrast, the combined defensive efficacy of both mutualist species reduced the volume of coral tissue lost by 73%, significantly more than the 38% reduction expected from independent defensive efforts, suggesting the existence of a cooperative synergy in defensive behaviors of 'guard' crustaceans. These emergent 'multiple defender effects' are statistically and ecologically analogous to the emergent concept of 'multiple predator effects' known from the predation literature.     

Therapeutic blockade of transforming growth factor beta fails to promote clearance of a persistent viral infection

Persistent viral infections often overburden the immune system and are a major cause of disease in humans. During many persistent infections, antiviral T cells are maintained in a state of immune exhaustion characterized by diminished effector and helper functions. In mammalian systems, an extensive immune regulatory network exists to limit unwanted, potentially fatal immunopathology by inducing T cell exhaustion. However, this regulatory network at times overprotects the host and fosters viral persistence by severely dampening adaptive immune responsiveness. Importantly, recent studies have shown that T cell exhaustion is mediated in part by host immunoregulatory pathways (e.g., programmed death 1 [PD-1], interleukin 10 [IL-10]) and that therapeutic blockade of these pathways either before or during persistent infection can promote viral clearance. Transforming growth factor beta (TGF-β) is another immunosuppressive cytokine known to impede both self- and tumor-specific T cells, but its role in regulating antiviral immunity is not entirely understood. In this study, we inhibited TGF-β with three potent antagonists to determine whether neutralization of this regulatory molecule is a viable approach to control a persistent viral infection. Our results revealed that these inhibitors modestly elevate the number of antiviral T cells following infection with a persistent variant of lymphocytic choriomeningitis virus (LCMV) but have no impact on viral clearance. These data suggest that therapeutic neutralization of TGF-β is not an efficacious means to promote clearance of a persistent viral infection.     

Gluconeogenesis is associated with high rates of tricarboxylic acid and pyruvate cycling in fasting northern elephant seals

Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ～95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.