Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy.

Mining of Phospholipase D (PLD) with high activity and stability has attracted strong interest for investigation. A novel PLD from marine Moritella sp. JT01 (MsPLD) was biochemically and structurally characterized in our previous study; however, the short half-life time (t1/2) under its optimum reaction temperature seriously hampered its further applications. Herein, the disulfide bond engineering strategy was applied to improve its thermostability. Compared with wild-type MsPLD, mutant S148C-T206C/D225C-A328C with the addition of two disulfide bonds exhibited a 3.1-fold t1/2 at 35 °C and a 5.7 °C increase in melting temperature (Tm). Unexpectedly, its specific activity and catalytic efficiency (kcat/Km) also increased by 22.7% and 36.5%, respectively. The enhanced activity might be attributed to an increase in the activation entropy by displacing more water molecules by the transition state. The results of molecular dynamics simulations (MD) revealed that the introduction of double disulfide bonds rigidified the global structure of the mutant, which might cause the enhanced thermostability. Finally, the synthesis capacity of the mutant to synthesize phosphatidic acid (PA) was evaluated. The conversion rate of PA reached about 80% after 6 h reaction with wild-type MsPLD but reached 78% after 2 h with mutant S148C-T206C/D225C-A328C, which significantly reduced the time needed for the reaction to reach equilibrium. The present results pave the way for further application of MsPLD in the food and pharmaceutical industries.

A New Phospholipase D from Moritella sp. JT01: Biochemical Characterization, Crystallization and Application in the Synthesis of Phosphatidic Acid.

A new phospholipase D from marine Moritella sp. JT01 (MsPLD) was recombinantly expressed and biochemically characterized. The optimal reaction temperature and pH of MsPLD were determined to be 35 °C and 8.0. MsPLD was stable at a temperature lower than 35 °C, and the t1/2 at 4 °C was 41 days. The crystal structure of apo-MsPLD was resolved and the functions of a unique extra loop segment on the enzyme activity were characterized. The results indicated that a direct deletion or fastening of the extra loop segment by introducing disulfide bonds both resulted in a complete loss of its activity. The results of the maximum insertion pressure indicated that the deletion of the extra loop segment significantly decreased MsPLD's interfacial binding properties to phospholipid monolayers. Finally, MsPLD was applied to the synthesis of phosphatidic acid by using a biphasic reaction system. Under optimal reaction conditions, the conversion rate of phosphatidic acid reached 86%. The present research provides a foundation for revealing the structural-functional relationship of this enzyme.

Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket.

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure-function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between ß9 and α9 (aa228-246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272-275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between ß11 and ß12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.

[Migration Characteristics of Manganese During Rainfall Events and Its Impacts on Water Quality in a Drinking Water Source Reservoir].

In view of the problem of excessive manganese concentrations in the Xi'an Jinpen Reservoir during the flood season in 2017, the vertical distribution of manganese in density currents and its occurrence pattern were monitored at multiple monitoring sections along the upstream reaches to the main basin. The influences of density currents plunging into the reservoir on the migration and transformation of Mn were studied, and sedimentation, output, and deposition of manganese in the reservoir water were also specifically estimated during a single, typical storm runoff process. Devices for avoiding high turbidity and high load inflows in rainfall events were proposed. The results showed that significant increases of total manganese were induced by high-turbidity inflows, which largely degraded water quality during rainfall events. From 12 to 14 October, 9.11 tons of total manganese were transported into the reservoir during a single rainfall event, and the pollution conditions were largely remitted by flood discharges with an output of 6.22 tons; thus, the net deposition (manganese) was 1.47 tons. The manganese content and morphological changes along the upper reaches of the reservoir indicated that soil erosion occurred during the continuous rainfall process, and this caused a large amount of particulate pollutants to flow into the water body with the runoff. More than 70% of the total manganese in the water was in the iron-manganese oxide bound state. Correlation analysis was conducted with particles of different particle size ranges, and granular manganese particle sizes were about 2-20 µm. The findings indicate that when flood discharges with turbidity currents occur, this can effectively reduce the load of pollutants and the safety risks of water.

[Sources and Distribution of Phosphorus in Sediments of the Jinpen Reservoir].

In order to explore the sources and distribution of phosphorus in sediments of a stratified water source reservoir, the total phosphorus (TP) content and distribution of phosphorus fractions in sedimentation particles and sediments of the Jinpen Reservoir of Xi'an were analyzed from March to November in 2017. The results showed that the TP content in the surface sediments of the Jinpen Reservoir was obviously affected by the deposition of particulate phosphorus (PP), and the correlation coefficient was 0.8775. Besides, this TP pool was also affected by the biogeochemistry of sediments. From June to August, algae in the Jinpen Reservoir propagated intensely, and a large number of dead algae were deposited on the bottom of the water body, which resulted in a type of endogenous pollution dominated by algae. The concentration of PP reached (753.51±17.11) mg·kg-1, and the content of TP increased, with NaOH-nrP as the main component. During the flood season of the Jinpen Reservoir from September to November, the runoff water carried a large amount of sediments with large pollutant loads, which resulted in increases of the concentration of PP in the water body. However, the TP content in the sediment per unit mass was relatively small. As a result, the TP content of surface sediments decreased, with inorganic Ca-P and rest-P as the main forms, which accounted for 55.8%-66.2% of the TP in sediment, and were influenced by particle sedimentation. The SRP, BD-P, and NaOH-srP, the most active fractions, underwent a series of transport and transformation processes under the changing environmental conditions (mainly redox conditions), and these forms were obviously affected by the biochemical processes in sediments and minimally affected by the sedimentation processes of the reservoir.

Controlling reduced iron and manganese in a drinking water reservoir by hypolimnetic aeration and artificial destratification.

The concentrations of iron (Fe) and manganese (Mn) in the water column have extremely important effects on the water quality of drinking water reservoirs; however, reservoirs often experience problematic Fe and Mn levels during seasonal stratification and rainfall events. Water-lifting aerators (WLAs) were deployed in the Jinpen Reservoir to control these issues with Fe and Mn at the source via bottom aeration and artificial destratification. In this study, variations of Fe and Mn concentrations in the water column, porewater, and sediments, were used to characterize behaviors of reduced Fe and Mn under the conditions of hypolimnetic aeration and artificial destratification during periods of hypolimnion hypoxia and rainfall events. The results showed that replenishing aquatic oxygen levels by aeration can effectively decrease the dissolved Fe and Mn in the water column thereby increasing the sedimentation rate and the diffusive flux of Fe and Mn at the sediment-water interface (SWI). The dissolved Fe was significantly chemically oxidized and the concentration remained relatively low in the water column during WLA operations, while dissolved Mn persistently accumulated in the near-sediment regions because of its complex kinetics. Our in situ profiles of labile Fe and Mn in the sediments demonstrated that the diffusive flux of Mn (JMn) was largely increased by the increased concentration gradient at the SWI, while the diffusive flux of Fe (JFe) decreased. The sediments were observed to rapidly become anoxic and release Fe and Mn after WLA deactivation; this emphasized the importance of appropriate operations linking the artificial and natural mixing periods to prevent SWI hypoxia and the release of reduced substances.

LPE-1, an orally active pyrimidine derivative, inhibits growth and mobility of human esophageal cancers by targeting LSD1.

Histone lysine specific demethylase 1 (LSD1) plays an important role in epigenetic modifications, and aberrant expression of LSD1 predicts tumor progression and poor prognosis in human esophageal cancers. In this study, a series of LSD1 inhibitors were synthesized and proved to be highly potent against human esophageal squamous cell carcinoma (ESCC). Our data showed that these LSD1 inhibitors selectively suppressed the viability of esophageal cancer cell line (EC-109) bearing overexpressed LSD1. Among these, compound LPE-1 (LSD1 IC50=0.336±0.003µM) significantly suppressed proliferation, induced apoptosis, arrested cell cycle of EC109 cells at G2/M phase, and caused changes of the associated protein markers correspondingly. We also found that compound LPE-1 potently inhibited the migration and invasion of EC-109 cells. Docking studies showed that the cyano group formed hydrogen bonds with Val811 and Thr810. Additionally, the thiophene moiety formed arene-H interaction with Trp761 residue. In vivo studies showed that compound LPE-1 inhibited tumor growth of xenograft models bearing EC-109 without obvious toxicity. Collectively, our findings indicate that LSD1 may be a potential therapeutic target in ESCC, and compound LPE-1 could serve as a lead compound for further development for anti-ESCC drug discovery.

Efficacy and safety of AIR inhaled insulin compared to insulin lispro in patients with type 1 diabetes mellitus in a 6-month, randomized, noninferiority trial.

BACKGROUND: Patients with type 1 diabetes may prefer features of AIR inhaled insulin (developed by Alkermes, Inc. [Cambridge, MA] and Eli Lilly and Company [Indianapolis, IN]; AIR is a registered trademark of Alkermes, Inc.) over insulin injection, but the two methods need to be compared for efficacy and safety. METHODS: This multicenter, 6-month, parallel-group, noninferiority trial had 500 patients with type 1 diabetes randomized to morning doses of basal insulin glargine plus either preprandial injectable insulin lispro or preprandial AIR insulin. We hypothesized that AIR insulin is noninferior (upper bound of the 95% confidence interval < or = 0.4%) to insulin lispro for change-from-baseline hemoglobin A1C (A1C). RESULTS: Baseline A1C was 7.95 +/- 0.08% for both groups. At end point, A1C was lower with insulin lispro than with AIR insulin by 0.27% (95% confidence interval 0.11, 0.43; P< 0.001). Noninferiority of AIR insulin to insulin lispro was not demonstrated, but similar percentages of patients in each group achieved A1C <7.0% (P = 0.448). Overall daily blood glucose was similar between groups at baseline (P = 0.879) and end point (P = 0.161). Two-hour postprandial blood glucose change from baseline was significantly (P < 0.001) higher with AIR insulin (20.77 +/- 4.33 mg/dL at 3 months and 15.85 +/- 3.08 mg/dL at end point) than with insulin lispro (3.29 +/- 4.14 mg/dL at 3 months and 1.67 +/- 2.91 mg/dL at end point). Overall hypoglycemia was similar between treatment groups (P = 0.355). The AIR insulin group had greater decrease in diffusing capacity of the lung for carbon monoxide at end point (P = 0.020) and greater incidence of cough (P = 0.024) and dyspnea (P = 0.030). Body weight decreased in the AIR insulin group and increased in the insulin lispro group. CONCLUSIONS: Insulin lispro provided lower A1C than AIR insulin, but the difference may not be clinically relevant.