PAOX1 expression in mixed-substrate continuous cultures of Komagataella phaffii (Pichia pastoris) is completely determined by methanol uptake regardless of the secondary carbon source: a simplifying principle for predicting recombinant protein production.

The expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional "secondary" carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses PAOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and AOX1 over a wide range of dilution rates (0.02-0.20 h-1) in continuous cultures of the Mut+ strain fed with methanol, methanol + glycerol, and methanol + sorbitol. We find that when the specific AOX1 expression and methanol uptake rates for each of the three feeds are plotted against each other, they collapse into a single hyperbolic curve. The specific AOX1 expression rate is therefore completely determined by the specific methanol uptake rate regardless of the existence (present/absent) and type (repressing/non-repressing) of the secondary carbon source. In particular, cultures fed with methanol + glycerol and methanol + sorbitol that consume methanol at equal rates also express the protein at equal rates and levels. Now, it turns out that the simple unstructured model developed by Egli and co-workers can predict the specific methanol uptake rates of single- and mixed-substrate cultures over a wide range of dilution rates and feed concentrations. By combining this model with our data, we derive simple formulas that predicts the protein expression rates and levels of single- and mixed-substrate cultures over a wide range of conditions.

Research on Parameters of Wire-Filling Laser Welding and Quenching Process for Joints Microstructure and Mechanical Property of BR1500HS Steel

With the aim to investigate the effect of parameter and quenching process on the joint of hot stamping steel by laser welding, the BR1500HS boron steel was welded by filling-wire laser welding with ER70-G welding wire under different parameters. The welded specimens were heated to 900℃ and held for 5min before water quenching. The universal material test machine, Optical micro-scope, Vickers hardness tester, scanning electron microscope and electron backscatter diffraction (EBSD) were used to characterize. The results showed that the macroscopic morphology of fusion zone (FZ) becomes from funnelform to hyperbolic curve shape when heat input increases and from hyperbolic curve shape to funnelform when wire-feed speed increases. The strength after quenching is more than 1557Mpa at heat input of 1040J/cm, wire feeding speed of 1.6m/min~1.8m/min and welding speed of 1.5m/min. EBSD test showed that the FZ and fine grain zone (FGZ) have more retained austenite (RA) than coarse grain zone (CGZ) before quenching and RA in FZ and heat affect zone (HAZ) decreased and distributed uniformed after quenching. The grain diameter in FZ distribute unevenly, with the maximum grain diameter larger than 40μm before quenching. After quenching, the grain diameter of FZ, HAZ and BM is more even and coarse grains in the FZ was refined. Before quenching, the microhardness of FZ and HAZ is of 450HV~500HV at different heat input and wire-feed speed and all region of joint keeps at 450HV~550HV after quenching. Most dimple and little river pattern in the joint fracture mor-phology before quenching indicates a well plasticity and most cleavage facet is observed after quenching due to the joint combine with martensite.

Shallow Imaging of Gas and Hydrate Using the Deep-towed ACS Data in Joetsu Basin, Niigata, Japan

<p>The deep-towed Autonomous Continuous Seismic (ACS) is a deep-towed marine seismic acquisition method. The ACS utilizes high frequency seismic source (ranging from 700 Hz to 2300 Hz) and multi-channel receivers that both source and receivers can be located close the seafloor. Moreover, the ACS is suitable to obtain high-resolution image of shallow geological structures. Since ACS data acquisition can be operated near the seafloor, the ocean (strong) current makes the position of both receivers and sources irregular (unstable) and it is hard to measure the absolute depth of both receivers and sources. During data processing, the unstable depth of both sources and receivers not only make the recorded seismic reflection curve (hyperbolic curve) rugged, but also makes the velocity analysis process more difficult because the velocity semblance is not clear. In this study, we propose a processing scheme to solve the unstable source–receiver position problem and thus to construct an accurate final stack profile (Hutapea et al., 2020 doi:10.1016/j.jngse.2020.103573). We used deep-towed ACS data acquired in the Joetsu Basin in Niigata, Japan, where hydrocarbon features in the form of gas chimneys, gas hydrate, and free gas have been observed. Furthermore, sidelobes in the ACS source signature defocus the source wavelet and decrease the bandwidth frequency content. We designed a filter to focus the source signature. Our proposed approach considerably improved the quality of bandwidth frequency of the source signature and the final stacked profile. Even though depth information was not available for all receivers, the velocity semblance was well focused. Our seismic attribute analyses for the final stack section shows that free gas accumulations are characterized by low reflection amplitude and an unstable frequency component, and that hydrate close to the seafloor can be identified by its high reflection amplitude.</p>

Isolation and Characterization of Leishmanial Adenine Aminohydrolase as a Drug target

Background:: Search for new drug targets is becoming imperative these days given that marketed chemotherapeutic drugs have lost their efficacy against harmful agents because of adaptability to climatic changes and co-evolving vectors to new hosts. In the wake of such challenge prominence of biochemical studies is increasing by way of exploring selective enzymes and investigating their structural and functional properties through biochemical kinetic parameter Km for the application of IC50 using designed drugs. Recently discovered Adenine Aminohydrolase [EC 3.5.4.2) in Leishmania has been found to be absent in mammalian purine salvage pathway and thus considered as a promising drug target against infectious agents. Objective:: The objective of this study is to isolate and characterize AAH by learning its kinetic mode of action using preferred substrate Adenine and additives estimated through expected product formation Hypoxanthine. Bioassays designed to measure exact Enzyme kinetic parameter Km value through establishing hyperbolic curve of enzyme reaction with the use of exact values of cellular quantities for IC50 application under experimental conditions devised by presteady state approach for SSA validity. Methods:: Following saturation kinetic, the plot of hyperbolic equilibrium curve developed using initial rates of product formation as a function of [Si] through forward shift under circumstance dG0 the system allows product and reactant favored reactions in relation to[Ef]1≈[E=KM] until complete saturation and estimates Km and Vmax of enzyme system under applied conditions. M-M equation used to assess experimental initial rate data for estimation of Km on excel using Solver and nonlinear least square coefficient correlation “R2”using logarithmic equation for nonlinear curve assessment. Results:: UV/Vis spectrophotometer selectively analyzed reacting components confirming Enzyme characteristic reaction constant Km equal toi15.0 ± 2 μ mol acquired from the Hyperbolic curve developed through use of exact [Si] ranges at selected parameter Km and Vmax. The curve assessed by Michaelis Menten equation provide Km value=14.99 μmol and non-linear least square coefficient correlation “R2” value equal to 0.9895,.along with that optimized lysis buffer formulation. In the docked complexes, the interactive amino acids identified were MSE441, ALA 364, GLN363, MSE518, VAL362, GLY517, ASP538, ALA445, TYR521, and TYR444. 2D interactions revealed hydrophobic and alkyl interactions at non-competitive binding site of the enzyme and therefore recommended as a potential inhibitors against 3ICS protein. Conclusion:: This study encourages biochemical analysis of the novel enzymes with the use of presteady state rationale in association with the computational tools as an effective way of designing drugs in short time against selective enzymes to meet the current challenge efficiently.

On a degenerate hyperbolic problem for the 3-D steady full Euler equations with axial-symmetry

The transonic channel flow problem is one of the most important problems in mathematical fluid dynamics. The structure of solutions near the sonic curve is a key part of the whole transonic flow problem. This paper constructs a local classical hyperbolic solution for the 3-D axisymmetric steady compressible full Euler equations with boundary data given on the degenerate hyperbolic curve. By introducing a novel set of dependent and independent variables, we use the idea of characteristic decomposition to transform the axisymmetric Euler equations as a new system which has explicitly singularity-regularity structures. We first establish a local classical solution for the new system in a weighted metric space and then convert the solution in terms of the original variables.

Computation Method for the Settlement of a Vertically Loaded Pile in Sloping Ground

Based on the hyperbolic curve tangent modulus method and the wedge stress theory, this paper proposes a calculation method for the settlement of a vertically loaded single pile in sloping ground. By establishing the relationship between the initial tangent modulus and the compression modulus of the slope soil, the tangent modulus of the slope soil is obtained combining with Mindlin-Geddes solution and Hansen formula, and the solution of the pile settlement in sloping ground is derived. Then, a series of numerical analyses are carried out to examine the feasibility of the proposed method. Finally, the effect of parameters is discussed in detail, including the slope angle, the distance of the pile from the slope crest, and the soil properties around the pile. The results show that the pile settlement will increase by increasing the slope angle or decreasing the distance of the pile from the slope crest, and the effect of the two parameters on the pile settlement is coupling-related. Besides, the compression modulus, cohesion, and internal friction angle of the soil around the pile are negatively related to the pile settlement and it is found that the compression modulus of the soil is the more influential parameter.

Force production of human cytoplasmic dynein is limited by its processivity

Cytoplasmic dynein is a highly complex motor protein that generates forces toward the minus end of microtubules. Using optical tweezers, we demonstrate that the low processivity (ability to take multiple steps before dissociating) of human dynein limits its force generation due to premature microtubule dissociation. Using a high trap stiffness whereby the motor achieves greater force per step, we reveal that the motor’s true maximal force (“stall force”) is ~2 pN. Furthermore, an average force versus trap stiffness plot yields a hyperbolic curve that plateaus at the stall force. We derive an analytical equation that accurately describes this curve, predicting both stall force and zero-load processivity. This theoretical model describes the behavior of a kinesin motor under low-processivity conditions. Our work clarifies the true stall force and processivity of human dynein and provides a new paradigm for understanding and analyzing molecular motor force generation for weakly processive motors.