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We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis-Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter K m and a 7-fold decrease in k cat These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

More accurate characterization and prediction of the in-situ distribution of fracturing fluid in fractured reservoirs are needed for enhancing well productivity. In this study, an implicit-sequentially coupled flow/geomechanics simulator incorporating an efficient discrete fracture model is developed to model fluid distribution and recovery performance of ultra-low permeability gas reservoirs. The finite-volume and finite-element methods are used for space discretization of the flow and geomechanics equations, respectively, while the backward Euler method is employed for time discretization. The flow and geomechanics equations are solved sequentially based on fixed-stress splitting. An efficient discrete-fracture model is used to explicitly model the fractured system. Flexible unstructured gridding is employed to model arbitrarily-oriented fractures. The interrelations among pore volume, permeability and geomechanical conditions are considered dynamically using two-way coupled flow and geomechanics computations. The geometry of fracture (networks) due to hydraulic fracturing has significant impacts on the fracturing fluid recovery efficiency and ensuing fluid distribution. Under the same injection volume, the fracturing fluid recovery is higher when the fracture geometry is planar. Fluid recovery is relatively lower whenever natural fractures are activated during fracturing treatments; flowback time is also shortened when complex fracture network with enlarged fracture interface is present. Fracturing fluid in hydraulic fractures may leak off into the natural fractures and subsequently imbibes into the surrounding matrix due to capillarity effects. The fracturing fluid recovery and in-situ fluid distribution are sensitive to the shut-in duration and fracture closure behavior. This study analyzes the coupled flow-geomechanical responses of fractured gas reservoirs during the post-fracturing periods. Understanding the fate of the fracturing fluid can provide insights on, to some extent, the stimulated fracture volume, size of the water invasion zone, and efficiency of the fracturing design. The simulation predictions can also provide more accurate initial reservoir conditions (e.g. distributions of different phases and pressure) for long-term well performance estimation.

The V27A mutation confers adamantane resistance on the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to determine structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to that of the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics simulation of the M2(22-46) V27A-spiro-adamantyl amine complex predicts with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2(22-46) V27A complex.

In order to study the effect of fermentation with a mixed starter culture of lactic acid bacteria on the quality of multigrain dough and steamed bread, yellow pea flour, naked oat flour, gluten flour and oat β-glucan (OBG) were mixed together to prepare dough and steamed bread with a direct vat set (DVS) mixed starter?culture of lactic acid bacteria. The acidifying capacity of lactic acid bacteria in dough was investigated, and the contents of OBG, resistant starch (RS) and free amino acids and the microstructure of dough were analyzed. Also, the quality and nutritional characteristics of steamed multigrain bread and steamed wheat bread before and after fermentation were compared. The results showed that lactic acid bacteria had strong acidifying capacity in fermented multigrain dough, and the contents of lactic acid and acetic acid were 24.85 and 8.98 mg/mL, respectively, after 24 hours of fermentation. Compared with unfermented multigrain dough, the OBG content in lactic acid bacteria fermented multigrain dough decreased by 32.56%, the RS content increased by 32.88%, the total amount of free amino acids increased by 1.46 times, the amino acid composition pattern was better, proteins and cellulose were partially degraded, and the gluten network was more compact and continuous. In terms of nutrition, the dietary fiber content of steamed multigrain bread was higher than 6% both before and after lactic acid bacteria fermentation, and the protein content was 40.35% and 38.38% in steamed unfermented and fermented multigrain bread, respectively, indicating that steamed multigrain bread is rich in dietary fiber and protein. Its dietary fiber and protein contents were higher than those of steamed wheat bread. Lactic acid bacteria fermented multigrain steamed bread had higher in vitro protein digestibility and specific volume, and the overall sensory score of steamed lactic acid bacteria fermented multigrain bread was significantly higher than that of steamed unfermented multigrain bread, and the overall acceptability was higher.

Using multi-walled carbon nanotubes (MWCNTs) as a novel sorbent and a new special anionic chromatographic column for pesticide residues, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous direct determination of glufosinate ammonium and its metabolites 3-methylphosphino-propionic acid and N-acetylglufosinate ammonium in milk. The samples were extracted with methanol, cleaned up using MWCNTs, and degreased by high-speed centrifugation at low temperature. The analytes were separated on an Anionic Polar Pesticide (2.1 mm × 100 mm, 5 μm) column using a mobile phase containing 0.9% formic acid solution (A) and 0.9% formic acid solution-acetonitrile (B). The instrument was operated in the negative ion scanning mode with multiple reaction monitoring. Glufosinate ammonium was quantified by an internal standard method, and its two metabolites by an external standard method. The calibration curves for glufosinate ammonium and its two metabolites were linear in the concentration range of 1–50, 2–50 and 2–50 μg/L with correlation coefficient (R2) greater than 0.99. For these compounds, the limits of detection were 2.5, 5.0 and 5.0 μg/kg, and the limits of quantification were 5.0, 10.0 and 10.0 μg/kg, respectively. The recoveries ranged from 79.35% to 101.80%, with relative standard deviation (RSD) between 1.15% and 8.63%. The proposed method is characterized by simple pretreatment, stable chromatographic conditions, low impurity interference, high sensitivity and good accuracy, and is suitable for the determination of glufosinate ammonium and its metabolite residues in milk.