Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM-1 s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM-1 s-1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

Designing a novel (R)-ω-transaminase for asymmetric synthesis of sitagliptin intermediate via motif swapping and semi-rational design.

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTAM8, which exhibited 79.2-fold higher activity than the chimeric scaffold MwTAMc. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates.

[Variation Characteristics and Potential Sources of the Mt. Haituo Aerosol Chemical Composition in Different Pollution Processes During Winter in Beijing, China].

In order to investigate the chemical composition and source apportionment of aerosols during winter in the Beijing-Tianjin-Heibei region, the particular matter (PM) and aerosol chemical composition at Mt. Haituo were observed by using a GRIMM 180, a single-particle soot photometer (SP2), and a high-resolution time-of-flight aerosol mass spectrometer (HR-TOF-AMS) from December 28, 2020 to February 3, 2021. Combining these observations with meteorological data and the HYSPLIT model, we calculated the potential source contribution factor (PSCF) and concentration weighted trajectory (CWT) and analyzed the temporal evolution and potential sources apportionment of PM and aerosol chemical composition under different pollution processes. The results showed that the dust storm process mainly affected PM10 and PM2.5 in Mt. Haituo during the winter and had a small impact on PM1; by contrast, haze pollution mainly affected PM1. Chemical components of aerosol accounted for 85.0% and 73.4% of PM1 on clean and haze days, respectively, but only 47.4% of PM1 in dust storm processes. NO3- was the chemical component with the largest mass concentration in haze, accounting for 25.2% of PM1; black carbon (BC) had the largest mass concentration on clean and dust storm days, accounting for 24.1% and 12.8% of PM1, respectively. The median diameters of BC were 209.7, 207.5, and 204.7 nm on clean, dust storm, and haze days, respectively. Dp/Dc was 2.15 in haze pollution, which was 1.38 and 1.39 times that on dust storm and clean days, respectively. Diurnal variations in PM and aerosol chemical components were different during the different processes. PM10 and PM2.5had high mass concentrations at night and low mass concentrations during the daytime on clean and dust storm days and had a unimodal distribution with a peak at 14:00 in haze. Diurnal variations in chemical composition had a unimodal distribution on clean days and a bimodal distribution on dust storm and haze days. The chemical compositions of the BC coating layer were different under different processes. The coating layers of BC were mainly NH4NO3, (NH4)2SO4, and organic matter on the clean, dust storm, and haze days, respectively. The distribution of potential sources of PM1 and its chemical components were different under different processes. The high-value area of the potential sources was mainly concentrated in the Beijing-Baoding-Shijiazhuang-Yangquan area in the southwestern portion of the site during dust storms and was mainly concentrated in Yanqing, Huailai, and Changping in the areas around the site during haze.

[Evolution and Potential Source Apportionment of Atmospheric Pollutants of Two Heavy Haze Episodes During the COVID-19 Lockdown in Beijing, China].

To control the spread of the 2019 novel coronavirus(COVID-19), China imposed rigorous restrictions, which resulted in great reductions in pollutant emissions. However, two heavy haze pollution episodes still occurred in Beijing. In this study, we use the air pollutants, aerosol number concentration, and meteorological elements data in Beijing, combined with the HYSPLIT model, to calculate the potential source contribution factor(PSCF) and concentration weight trajectory(CWT), and analyze the characteristics of evolution and potential source apportionment of atmospheric pollutants during the two episodes. The COVID-19 lockdown restrictions had great impacts on the diurnal variations of PM2.5 and black carbon(BC), while small impacts on the diurnal variations of CO, NO2, SO2, and O3. The primary pollutant was PM2.5 during the two haze pollution episodes, and the haze1 episode was mainly local pollution, while haze 2 was mainly local and external transportation pollution. The spectrum of aerosol number concentration was unimodal under different processes, with the peak located at 0.3 µm. During the haze episodes, the number concentration in the size range of 0.2-0.5 µm increased 3.3-13.6 times that of the clean days. The mass concentration contributions of BCliquid to BC in different processes were 64.8%-85.1%. This mass concentration of BCliquid ranked in the order of haze 1(5.04 µg·m-3) >haze 1(3.20 µg·m-3) >clean day(before COVID-19) (2.31 µg·m-3) >clean day(COVID-19) (0.76 µg·m-3). The characteristics of PSCF and CWT distribution of PM2.5 and BC were different in different processes. The PSCF high value areas of PM2.5 on the clean day(before COVID-19) and the clean day(COVID-19) were mainly distributed in the southwest and western of Beijing, and the weight concentration exceeded 30 µg·m-3. The PSCF high value areas of PM2.5 during haze 1 and haze 2 were mainly distributed in Beijing and its surrounding areas and southwestern, when the weight concentration exceeded 90 µg·m-3. The PSCF high value areas of BC were mainly distributed in Beijing and its surrounding areas on clean days(before COVID-19), clean days(COVID-19) and haze 1, with weighted concentrations exceeding 2.4, 0.9 and, 4.5 µg·m-3, respectively. The PSCF high value areas of BC on haze 2 was distributed in the southwest of Beijing, and the weight concentration exceeded 5 µg·m-3.

Internalized gold nanoparticles do not affect the osteogenesis and apoptosis of MG63 osteoblast-like cells: a quantitative, in vitro study.

The long-term toxicity effects of gold nanoparticles (GNPs) on the proliferation and differentiation of a progenitor cell line, MG63 osteoblast-like cells, was investigated. These cells were treated for 20 hours with two media that contained 10 nm GNPs at concentrations of 1 ppm and 10 ppm. The mitosis of the GNP-treated MG63 was observed after at least 21 hours using dark-field and fluorescence microscopy. The TEM, LSCM and dark-field hyperspectral images indicated that the late endosomes in cells that contained aggregated GNPs were caused by vesicle fusion. Subsequently, after 21 days of being cultured in fresh medium, the specific nodule-like phenotypes and bone-associated gene expression of the treated MG63 cells exhibited the same behaviors as those of the control group. Statistically, after 21 days, the viability of the treated cells was identical to that of the untreated ones. During the cell death program analysis, the apoptosis and necrosis percentages of cells treated for 8 or fewer days were also observed to exhibit no significant difference with those of the untreated cells. In summary, our experiments show that the long-term toxicity of GNPs on the osteogenetic differentiation of MG63 is low. In addition, because of their low toxicity and non-biodegradability, GNPs can potentially be used as biomarkers for the long-term optical observation of the differentiation of progenitor or stem cells based on their plasmonic light-scattering properties.

[Temporal and spatial distribution of ozone concentration by aircraft sounding over Beijing].

Based on the aircraft sounding volume fraction concentration data of ozone (O3), nitrogen oxides (NO, NO2) and other data in Beijing from 2007 to 2010, temporal and spatial evolution of ozone concentration from the ground surface to 3.5 km altitude were studied. Results show that: (1) Vertical profiles of monthly average O3 concentration were in good agreement, with increasing altitude, the concentrations were first increased and then decreased, and then remained almost constant, and there was a clear dividing line at 1.5 km altitude, the vertical gradient of the O3 concentration changed greatly below which, there were O3 high-value areas, which were influenced by human activities near the ground; the change of vertical gradient of O3 concentration was significantly reduced above 1.5 km altitude, this space was above the mixing layer, where the air mass movement was less affected by underlying surface, and the advection-diffusion played a crucial role in the local accumulation process of air pollutants. (2) Changes of O3 concentration showed clear seasonal characteristics, O3 concentration was lower in spring and autumn, but higher in summer. In the months studied, no significant difference in monthly average O3 concentration from July to September was detected (P > 0.05), but there was significant difference in other months (P < 0.01). (3) In summer days (daytime), the variations in the vertical profiles of hourly O3 concentration were consistent with those of the monthly O3 concentration. The O3 concentration was lower near the surface within 1.5 km in the morning (09:00-10:00), and higher in the afternoon (15:00-16:00), with the maximum discrepancy of about 60 x 10(-9) in the same altitude; there was minor difference in O3 concentration in altitude range of 1.5-3.5 km, generally fluctuating among 70 x 10(-9) -80 x 10(-9). (4) For the regional distribution of O3 concentration, higher concentration within 0-2 km appeared near the Fourth Ring Road of city center and the surrounding areas, the main reasons for this distribution might be the presence of many strong sources of pollution emissions and low sink flow near the ground; within 2-4 km, in addition to the urban area of Beijing, higher O3 concentration areas were found in the north, the south-east (Beijing-Tianjin direction), the south-west (Beijing-Baoding direction). (5) There were significant correlation between O3 and NO, NO2 and NO2/NO within 0-3.5 km, O3 was negatively related with both NO and NO2, but positively correlated with the NO2/NO ratio.

Supratherapeutic anticoagulation from low-molecular-weight heparin in lung transplant recipients.

BACKGROUND: Venous thromboembolism (VTE) is common after lung transplantation. Enoxaparin is an approved therapy for VTE and anti-factor Xa level can be used to monitor enoxaparin activity. Some studies have demonstrated elevated anti-factor Xa levels are associated with an increased risk of hemorrhage. Having identified a high incidence of supratherapeutic anti-factor Xa levels in lung transplant recipients, we aimed to elucidate the relationship between enoxaparin dose and anti-factor Xa level in this patient population. METHODS: We identified post-lung transplantation patients with VTE receiving therapeutic enoxaparin who had anti-factor Xa level measured. Standard enoxaparin dosing was defined as 0.9 to 1.1 mg/kg. After identifying a high incidence of supratherapeutic anti-factor Xa levels, we implemented "non-standard" dosing of 0.8 mg/kg. Multivariate linear regression analysis was used to examine the association between enoxaparin dose and anti-factor Xa level; age, body mass index (BMI) and creatinine clearance were included as covariates. RESULTS: In the cohort, 18 patients received standard and 8 patients received non-standard enoxaparin dosing. Twelve of 18 patients (67%; 95% confidence interval [CI]: 43% to 91%) receiving standard dosing had supratherapeutic anti-factor Xa levels vs 0 of 8 patients (0%; 95% CI: 0% to 37%) receiving lower non-standard dosing (p = 0.002). Anti-factor Xa levels were significantly different between the two groups; the mean anti-factor Xa level was 1.3 IU/ml (95% CI: 1.06 to 1.53) in the standard group vs 0.79 IU/ml (95% CI: 0.67 to 0.91) in the non-standard group (p = 0.008). After controlling for covariates, for each 0.1-mg/kg increase in enoxaparin, the mean anti-factor Xa level increased by 0.18 IU/ml (95% CI: 0.05 to 0.31; p = 0.011; model r(2) = 0.53). CONCLUSIONS: Standard dosing of enoxaparin in lung transplant recipients is associated with a high incidence of supratherapeutic anti-Xa levels. Further study will be required to correlate this finding with risk of hemorrhage.

Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affect hexenal signaling in the Columbia and Landsberg erecta ecotypes of Arabidopsis.

The CYP74B2 gene in Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col) contains a 10-nucleotide deletion in its first exon that causes it to code for a truncated protein not containing the P450 signature typical of other CYP74B subfamily members. Compared to CYP74B2 transcripts in the Landsberg erecta (Ler) ecotype that code for full-length hydroperoxide lyase (HPL) protein, CYP74B2 transcripts in the Col ecotype accumulate at substantially reduced levels. Consistent with the nonfunctional HPL open reading frame in the Col ecotype, in vitro HPL activity analyses using either linoleic acid hydroperoxide or linolenic acid hydroperoxide as substrates show undetectable HPL activity in the Col ecotype and C6 volatile analyses using leaf homogenates show substantially reduced amounts of hexanal and no detectable trans-2-hexenal generated in the Col ecotype. P450-specific microarrays and full-genome oligoarrays have been used to identify the range of other transcripts expressed at different levels in these two ecotypes potentially as a result of these variations in HPL activity. Among the transcripts expressed at significantly lower levels in Col leaves are those coding for enzymes involved in the synthesis of C6 volatiles (LOX2, LOX3), jasmonates (OPR3, AOC), and aliphatic glucosinolates (CYP83A1, CYP79F1, AOP3). Two of the three transcripts coding for aliphatic glucosinolates (CYP83A1, AOP3) are also expressed at significantly lower levels in Col flowers.