Evolving ω-amine transaminase AtATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates.

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus (AtATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate (R)-(+)-1-(1-naphthyl)ethylamine [(R)-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 (AtATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to AtATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing (R)-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the AtATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level.

Arsenic Accumulation and Physiological Response of Three Leafy Vegetable Varieties to As Stress.

Arsenic (As) in leafy vegetables may harm humans. Herein, we assessed As accumulation in leafy vegetables and the associated physiological resistance mechanisms using soil pot and hydroponic experiments. Garland chrysanthemum (Chrysanthemum coronarium L.), spinach (Spinacia oleracea L.), and lettuce (Lactuca sativa L.) were tested, and the soil As safety threshold values of the tested leafy vegetables were 91.7, 76.2, and 80.7 mg kg−1, respectively, i.e., higher than the soil environmental quality standard of China. According to growth indicators and oxidative stress markers (malondialdehyde, the ratio of reduced glutathione to oxidized glutathione, and soluble protein), the order of As tolerance was: GC > SP > LE. The high tolerance of GC was due to the low transport factor of As from the roots to the shoots; the high activity of superoxide dismutase, glutathione peroxidase, and catalase; and the high content of phytochelatin in the roots. Results of this work shed light on the use of As-contaminated soils and plant tolerance of As stress.

[Effect of Film Mulching, Straw Retention, and Nitrogen Fertilization on the N2O and N2 Emission in a Winter Wheat Field].

In order to explore the characteristics of N2O emissions from winter wheat fields in the Loess Plateau under different farming methods and nitrogen levels, the dynamic changes in N2O emissions from rain-fed winter wheat fields were quantified using static box-gas chromatography. Winter wheat 'Xiaoyan22' was used as the material, and a two-factor split area design was adopted. The conventional tillage (CT), straw incorporated into soil (SM), and flat film mulching (FM) were assigned as the main plot, and three nitrogen fertilizer rates (no nitrogen fertilization, 20% nitrogen reduction (144 kg·hm-2), and conventional nitrogen application (180 kg·hm-2)) were assigned as a split plot. Taking CT as a control, the effects of FM and SM on soil N2O emissions under different nitrogen rates were assessed. Furthermore, the correlation between relevant environmental factors and N2O emission flux were analyzed, and N2 emissions were estimated using empirical formulas. The results showed the following:the N2O emissions from the soil of each nitrogen treatment occurred within 20 days, and N2O emission flux peaked within two weeks post-fertilization. The average N2O flux, the total N2O emissions, and the global warming potential of N2O were 1.92-22.75 µg·(m2·h)-1, 0.10-0.46 kg·hm-2, and 26.72-122.15 kg·hm-2, respectively. The N2O emission coefficient of fertilizer nitrogen was 0.05%-0.28%. The total N2 emissions ranged from 0.70-1.82 kg·hm-2. The N fertilization and film mulching significantly increased the N2O emission flux (P<0.05) and the cumulative N2O emissions (P<0.05); however, SM marginally reduced the total N2O emissions. The N2O emission coefficient and global warming potential of fertilizer nitrogen under FM were significantly higher than those under CT and SM (P<0.05). The N2O emissions without nitrogen treatment were only significantly positively correlated with soil water-filled pore spaces (WFPS) (P<0.05); the N2O emissions in the N fertilization condition were significantly positively correlated with WFPS, ω(NO3--N), ω(NH4+-N), and 0-5 cm soil layer temperature (P<0.05). Overall, under the condition of no fertilization, water was the main factor to control the nitrogen transformation and soil N2O emission; nevertheless, under the N fertilization condition, both nitrification and denitrification contributed to the N2O emissions in the rain-fed winter wheat fields. Film mulching practice and nitrogen application markedly increased the N2O emissions, fertilizer nitrogen emission coefficient, and global warming potential in the rain-fed winter wheat fields. Nonetheless, straw incorporated into the soil resulted in a marginal reduction in N2O emissions.

Essential thrombocythemia with non-ST-segment elevation myocardial infarction as the first manifestation: A case report.

BACKGROUND: We report a case of essential thrombocythemia (ET) in a 44-year-old male who exhibited non-ST-segment-elevation myocardial infarction (NSTEMI) as the first manifestation without known cardiovascular risk factors (CVRFs). For the first time, we reported a left main trifurcation lesion in NSTEMI caused by ET, including continuous stenosis lesions from the left main to the ostial left anterior descending (LAD) artery and an obvious thrombotic lesion in the ostial and proximal left circumflex (LCX) artery. There was 60% diffuse stenosis in the left main (LM) that extended to the ostial LAD, thrombosis of the ostial LAD and proximal LCX, and 90% stenosis in the proximal LCX. During the operation, thrombus aspiration was performed, but no obvious thrombus was aspirated. Performing the kissing balloon technique (KBT) in the LCX and LM unexpectedly increased the narrowness of the LAD. Then, the single-stent crossover technique, final kissing balloon technique and proximal optimization technique (POT) were performed. On the second day after percutaneous coronary intervention (PCI), the number of platelets (PLTs) still increased significantly to as high as 696 × 109/L. The bone marrow biopsy done later, together with JAK2 (exon 14) V617F mutation, confirms the diagnosis of ET. Hydroxyurea was administered to inhibit bone marrow proliferation to control the number of PLTs. CASE SUMMARY: A 44-year-old male patient went to a local hospital for treatment for intermittent chest pain occurring over 8 h. The examination at the local hospital revealed elevated cTnI and significantly elevated platelet. Then, he was diagnosed with acute myocardial infarction and transferred to our hospital for emergency interventional treatment by ambulance. During the operation, thrombus aspiration, the single-stent crossover technique, final kissing balloon technique and POT were performed. Dual antiplatelet therapy comprising aspirin and ticagrelor was used after PCI. Evidence of mutated JAK2 V617F and bone marrow biopsy shown the onset of ET. Together with JAK2 (exon 14) V617F mutation, ET was diagnosed according to the World Health Organization (WHO) diagnostic criteria, and the patient was placed on hydroxyurea. During the one-year postoperative period, repeated examinations showed a slight increase in PLTs, but the patient no longer had chest tightness, chest pain or bleeding or developed new thromboembolisms. CONCLUSION: Routine physical examinations and screenings are conducive to the early detection of ET, and the risk for thrombosis should be assessed. Then, active antiplatelet therapy and myelosuppression therapy should be used for high-risk ET patients.

[Effects of N and S application on grain filling characteristics and yield of winter wheat].

A field experiment was conducted on the Guanzhong Plain, China, to evaluate the effects of N and S application on characteristics of winter wheat (cv. Xiaoyan 22) grain filling and yield, using a central composite rotatable design with two factors nitrogen (N) and sulfur (S). The results showed that, with the combined application of N and S, grain filling followed a "S" curve which increased slowly at first, then quickly, and then slowly again. With 108 and 267 kg N x hm(-2), the grain fill duration, theoretical maximum grain yield, and average filling rate decreased as the S application rate increased. With 97.5 and 202.5 kg S x hm(-2), N application improved grain filling parameter values. With 187.5 kg N x hm(-2) or 150 kg S x hm(-2), the parameters firstly increased, reached a maximum, and then decreased as the S or N input increased. Grain filling rate increased for 25 days following anthesis, and then declined at a rate that varied among treatments. When the N input was > 187.5 kg x hm(-2) or the S input was > 150 kg x hm(-2), the grain filling rate decreased with increasing the S or N input. The results also indicated that combined application of N and S fertilizers at appropriate rates could increase the head density and grain yield. Using the regression equations, highest grain yields estimates (> or = 3753 kg x hm(-2)) were achieved with the combination of a high N rate (178.31-256.36 kg x hm(-2)) and a moderate S rate (131.95-167.65 kg x hm(-2)).

[Factors influencing ammonia volatilization in a winter wheat field with plastic film mulched ridges and unmulched furrows].

The objective of this experiment was to quantify ammonia volatilization from a winter wheat field with plastic film mulched-ridges and unmulched-furrows (PMRF). The trial was conducted during the 2010-2011 winter wheat growing season at Yangling, Shaanxi Province. Ammonia volatilization from the soil was measured using the closed-chamber method. The results indicated that NH3 emission losses ranged between (1.66 +/- 0.3) and (3.28 +/- 0.51) kg x hm(-2) in the PMRF treatment. In comparison, the NH3 emission loss was (4.68 +/- 0.35) kg x ha(-1) in the conventional tillage treatment (i. e., smooth soil surface). The PMRF treatment reduced NH3 emissions by 29.8 to 63.8% compared with the conventional treatment. The NH3 emission losses were equivalent to 1.9% of the applied N in the conventional practice treatment. In contrast, the losses were equivalent to only 0.3% to 0.8% of the applied N in the PMRF treatment. Ammonia emissions were greatest during the first two weeks after sowing. Emissions before winter accounted for 82% of total NH3 emission in the conventional practice treatment, but only 49% to 61% of the total NH3 emission in the PMRF treatment. The soil NH4+ -N content and the soil moisture content had direct effects on NH3 emission before winter in the conventional treatment. In thePMRF treatment, the soil NH4+ -N content had a direct effect on NH3 emission before winter, whereas soil surface temperature and soil moisture had indirect effects. Ammonia emissions after the greening stage were mainly influenced by the soil NH4+ -N content. Simulation results indicated that logarithmic functions best described cumulative NH3 emission in the PMRF + high N rate treatment and the conventional treatment. A linear function best described cumulative NH3 emission in the PMRF + low N rate treatment and the unfertilized treatment. In conclusion, the PMRF treatment can significantly reduce N losses from winter wheat fields by changing the spatial-temporal dynamics of soil NH3 volatilization.

[Effects of seeding rate and nitrogen fertilization rate on physiological traits of winter wheat after anthesis in a field with plastic mulched rides and unmulched furrows].

Taking winter wheat cultivar Xiaoyan 22 as test material, a field experiment with central composite rotatable design was conducted to investigate the effects of seeding rate and nitrogen (N) fertilization rate on the physiological traits of the cultivar after anthesis under ridge-furrow cultivation with plastic film mulching, aimed to harmonize the relationships between winter wheat population and its individuals and to fully use the advantages of ridge-furrow cultivation in dry land areas. With increasing N fertilization rate, the leaf area index and the flag leaf chlorophyll content and net photosynthesis rate after anthesis increased; with increasing seeding rate, the leaf area index increased first and was stable then at early and middle milking stages but decreased at later milking stage, the flag leaf chlorophyll content and net photosynthesis rate decreased, and the grain yield per plant increased after an initial decrease. Appropriate seeding rate could optimize the relationships between winter wheat population and its individuals, and optimal N fertilization rate benefited the improvement of winter wheat physiological traits after anthesis and the enhancement of winter wheat yield. Under our experimental condition, seeding rate 112.5 kg x hm(-2) plus N fertilization rate 180-222 kg N x hm(-2) is a scheme beneficial to optimize the relationships between winter wheat population and its individuals and to establish a good winter wheat community structure with a reasonable leaf area index after anthesis, higher flag leaf chlorophyll content and net photosynthesis rate, higher grain yield per plant, and high yield per unit area.

[Adsorption behavior and speciation transformation of Ag+ in phaeozem and red soil].

The batch equilibration experiments were carried out to study the adsorption characteristics of Ag+ and the effect of pH on the adsorption process in two typical Chinese soils, which the phaeozem from Northeast China and the red soil from Hunan province. The species transformation of Ag+ was investigated in the experiments using the sequential extraction method. The results showed that an adsorption quantity of Ag+ for both soils increased with increasing solutions initial pH. Adsorption quantity of Ag+ in the two soils also increased with increasing of initial Ag+ concentration in the range from 0 to 100 mg x L(-1), specifically, going up quickly in the concentration range of 0-20 mg x L(-1), but increased gently when Ag+ concentration exceeded 20 mg x L(-1). The Ag+ adsorption data of both soils were adequately described by Langmuir isotherm occurred in Ag+ sorption processes, their maximum adsorption quantities were 250 mg x kg(-1) the phaeozem soil and 88.5 mg x kg(-1) in the red soil, indicating a much higher adsorption capacity in northeast phaeozem soil than that of Hunan red soil. The two soils had similar adsorption kinetic characteristics, with respect to quickly reaching equilibrium within 30 minutes, but the adsorption rate of phaeozem soil was significantly greater than that of the red soil. The species analysis of Ag showed that exchangeable Ag and residual Ag increased in red soil, and metal-organic complex-bound Ag, exchangeable Ag, carbonate-bound Ag and residual Ag increased in phaeozem, respectively.