Engineered E. coli for D-pantothenic acid production with an acetolactate isomeroreductase mutant.

D-Pantothenic acid, as a momentous vitamin, is extensively applied to feed, medicine, cosmetics and other fields. However, there are still limitations to produce D-pantothenic acid by microbial fermentation at present. In this paper, we constructed a recombinant strain for D-pantothenic acid production by blocking the organic acid pathway, boosting pyruvate biosynthesis, relieving feedback inhibition of acetolactate synthase, improving glucose intake capacity, and modifying essential genes in the metabolic pathway. In addition, a new acetolactate isomeroreductase mutant V412A origin from Escherichia coli (EcAHAIR) encoded by ilvC was obtained to explore its substrate promiscuity. Compared with the wild type, the variant EcAHAIR-V412A has reduced steric hindrance and enhanced intermolecular forces, resulting in a high affinity for 2-acetolactate. Eventually, the fermentation production of the final strain DPAN19/trc-ilvCV412A reached 4.65 g/L, increased by 192.5% compared with strain DPA8 in shake flask cultivation and produced 62.82 g/L D-pantothenic acid in a 5 L bioreactor. The metabolic engineering strategies and enzyme modification approaches described in this paper provide a particular perspective for the bio-manufacturing of D-pantothenic acid, branched-chain amino acids and its derivates.

Rapid Atmospheric Reactions between Criegee Intermediates and Hypochlorous Acid.

Hypochlorous acid (HOCl) is a paramount compound in the atmosphere due to its significant contribution to both tropospheric oxidation capacity and ozone depletion. The main removal routes for HOCl are photolysis and the reaction with OH during the daytime, while these processes are unimportant during the nighttime. Here, we report the rapid reactions of Criegee intermediates (CH2OO and anti/syn-CH3CHOO) with HOCl by using high-level quantum chemical methods as the benchmark; their accuracy is close to coupled cluster theory with single, double, and triple excitations and quasiperturbative connected quadruple excitations with a complete basis limit by extrapolation [CCSDT(Q)/CBS]. Their rate constants have been calculated by using a dual-level strategy; this combines conventional transition state theory calculated at the benchmark level with variational transition state theory with small-curvature tunneling by a validated density functional method. We find that the introduction of the methyl group into Criegee intermediates not only affects their reactivities but also exerts a remarkable influence on anharmonicity. The calculated results uncover that anharmonicity increases the rate constants of CH2OO + HOCl by a factor of 18-5, while it is of minor importance in the anti/syn-CH3CHOO + HOCl reaction at 190-350 K. The present findings reveal that the loose transition state for anti-CH3CHOO and HOCl is a rate-determining step at 190-350 K. We also find that the reaction of Criegee intermediates with HOCl contributes significantly to the sink of HOCl during the nighttime in the atmosphere.

Kinetics of Sulfur Trioxide Reaction with Water Vapor to Form Atmospheric Sulfuric Acid.

Although experimental methods can be used to obtain the quantitative kinetics of atmospheric reactions, experimental data are often limited to a narrow temperature range. The reaction of SO3 with water vapor is important for elucidating the formation of sulfuric acid in the atmosphere; however, the kinetics is uncertain at low temperatures. Here, we calculate rate constants for reactions of sulfur trioxide with two water molecules. We consider two mechanisms: the SO3···H2O + H2O reaction and the SO3 + (H2O)2 reaction. We find that beyond-CCSD(T) contributions to the barrier heights are very large, and multidimensional tunneling, unusually large anharmonicity of high-frequency modes, and torsional anharmonicity are important for obtaining quantitative kinetics. We find that at lower temperatures, the formation of the termolecular precursor complexes, which is often neglected, is rate-limiting compared to passage through the tight transition states. Our calculations show that the SO3···H2O + H2O mechanism is more important than the SO3 + (H2O)2 mechanism at 5-50 km altitudes. We find that the rate ratio between SO3···H2O + H2O and SO3 + (H2O)2 is greater than 20 at altitudes between 10 and 35 km, where the concentration of SO3 is very high.

Quantitative kinetics for the atmospheric reactions of Criegee intermediates with acetonitrile.

Obtaining quantitative kinetics of Criegee intermediates is of paramount significance in the atmosphere. However, there are limited reports on the kinetics of Criegee intermediates. Here, by using our very recently developed dual-level strategy, we report the quantitative kinetics of the reaction of Criegee intermediates (CH2OO/anti-CH3CHOO/syn-CH3CHOO) with acetonitrile (CH3CN). The dual-level strategy combines post-CCSD(T) calculations for transition state theory with the validated M06CR/MG3S and M11-L/MG3S functional methods for direct dynamics calculations using canonical variational transition state theory with small-curvature tunneling to obtain recrossing effects and tunneling coefficients. We show that W3X-L//DF-CCSD(T)-F12b/jun-cc-pVDZ can be used to obtain quantitative enthalpies of activation at 0 K in the reactions of Criegee intermediates with CH3CN. We find that the CH2OO/anti-CH3CHOO/syn-CH3CHOO + CH3CN reactions only depend on temperature. Moreover, we also find that their rate constants are dominantly determined by the enthalpy of activation at 0 K and recrossing effects and tunneling are negligible. The present findings also show that the CH2OO/anti-CH3CHOO + CH3CN reactions have negative temperature dependence in the range of 190-350 K. In the atmosphere, we reveal that the reactions of CH2OO and anti-CH3CHOO with CH3CN are significant acetonitrile sinks, leading to the formation of N-formylacetamide and diacetamide. The present findings will be useful for obtaining quantitative kinetics of Criegee intermediates and understanding acetonitrile sinks.

Differences of Matrix Metalloproteinase 2 Expression between Left and Right Ventricles in Response to Nandrolone Decanoate and/or Swimming Training in Mice.

BACKGROUND: Matrix metalloproteinase (MMP)-2 plays an important role in the remodeling of left ventricles (LVs) and right ventricles (RVs). We investigated the differences of MMP-2 expression between LV and RV in response to nandrolone decanoate (ND), swimming training (ST), and combined ND and ST (NS) in mice, based on their structural, functional, and biochemical characteristics. METHODS: Totally 28 male C57B1 mice (6 weeks old; 20-23 g) were divided into four groups, including the control (n = 7), ND (n = 6), ST (n = 8), and NS (n = 7) groups. After respective treatments for 8 weeks, echocardiographic examination was used to assess the cardiac structure and function. Van Gieson stain was used to examine the fibrosis of LV and RV in response to different treatments, and Western blotting analysis was performed to explore different MMP-2 expressions between LV and RV in response to ND and/or ST. Analysis of variance was used for comparing the four groups. RESULTS: At 8 weeks, right ventricular dimension/body weight in the ND group was larger than the other three groups (F = 7.12, P < 0.05) according to the echocardiographic examination. Fibrosis induced by ND administration was increased more in RV (2.59%) than that in LV (2.21%). MMP-2 expression of the ND group in RV was significantly greater than the control and NS groups in RV and the corresponding ND group in LV. CONCLUSION: The experimental data support the hypothesis that ND administration induces greater MMP-2 expression increase in RV compared to LV, leading to consequent RV dilation.

Therapeutic effects of curcumin nanoemulsions on prostate cancer.

The therapeutic potential of curcumin (Cur) is hampered by its poor aqueous solubility and low bioavailability. The aim of this study was to determine whether Cur nanoemulsions enhance the efficacy of Cur against prostate cancer cells and increase the oral absorption of Cur. Cur nanoemulsions were developed using the self-microemulsifying method and characterized by their morphology, droplet size and zeta potential. The results showed that the cytotoxicity and cell uptake were considerably increased with Cur nanoemulsions compared to free Cur. Cur nanoemulsions exhibited a significantly prolonged biological activity and demonstrated better therapeutic efficacy than free Cur, as assessed by apoptosis and cell cycle studies. In situ single-pass perfusion studies demonstrated higher effective permeability coefficient and absorption rate constant for Cur nanoemulsions than for free Cur. Our study suggested that Cur nanoemulsions can be used as an effective drug delivery system to enhance the anticancer effect and oral bioavailability of Cur.

Therapeutic Effects of Curcumin Nanoemulsions on Prostate Cancer / 华中科技大学学报（医学）（英德文版）

The therapeutic potential of curcumin (Cur) is hampered by its poor aqueous solubility and low bioavailability.The aim of this study was to determine whether Cur nanoemulsions enhance the efficacy of Cur against prostate cancer cells and increase the oral absorption of Cur.Cur nanoemulsions were developed using the self-microemulsifying method and characterized by their morphology,droplet size and zeta potential.The results showed that the cytotoxicity and cell uptake were considerably increased with Cur nanoemulsions compared to free Cur.Cur nanoemulsions exhibited a significantly prolonged biological activity and demonstrated better therapeutic efficacy than free Cur,as assessed by apoptosis and cell cycle studies.In siru single-pass perfusion studies demonstrated higher effective permeability coefficient and absorption rate constant for Cur nanoemulsions than for free Cur.Our study suggested that Cur nanoemulsions can be used as an effective drug delivery system to enhance the anticancer effect and oral bioavailability of Cur.

[Effects of phytohormones on hairy root growth and tanshinone biosynthesis of Salvia miltiorrhiza].

OBJECTIVE: To study the effects of different phytohormones on the growth of hairy root and biosynthesis of tanshinone II A in Salvia miltiorrhiza. METHODS: Salvia miltiorrhiza hairy root was induced by organizational culture method. Tanshinone was extracted with ultrasonic method and detected by high performance liquid chromatography (HPLC). RESULTS: The phytohormone combination of GA3 and 6-BA at different concentrations adding to 1/2 MS medium had obviously inhibitory effect on the hairy root growth of Salvia miltiorrhiza. And the highest inhibition rate was up to 100% (hairy roots were death). However, the growth of hairy roots could be obviously promoted by the combination of KT (1.0 mg/L) and IBA (1.0 mg/L), increasing 3. 251 times than that of the control. Total tanshinone II A content in hairy roots bodies was the highest under the combination of 0.2 mg/L NAA and 3.0 mg/L 6-BA. 6-BA of 2.0 mg/L played a significant role in promoting tanshinone II A biosynthesis, and the tanshinone II A concentration was 3.012 times higher than that of the control. CONCLUSION: Different phytohormones and its combination adding to 1/2 MS medium has significant influence on the hairy root growth and the content of tanshinone II A in Salvia miltiorrhiza.

[Prenatal risk factors for neonatal asphyxia: how risk for each?].

OBJECTIVE: Neonatal asphyxia is the third leading cause of neonatal death and main cause of long-term neurodevelopmental handicap throughout the world. Prevention is more important than treatment. Most previous reports are limited to retrospective investigations of the relationships between some prenatal risk factors and low Apgar scores. This study was designed to prospectively investigate the relationship between prenatal risk factors and neonatal asphyxia and the influence of single or multiple risk factors on the incidence of neonatal asphyxia, and examine significant risk factors for neonatal asphyxia. METHODS: From April 2002 through October 2004, a total of 10 376 live-born newborns were enrolled. Forty-six prenatal risk factors were investigated. Neonatal asphyxia was diagnosed based on the following four items: 1. 1-min Apgar score or=0.10 and p<0.05 as significant. The OR and 95%CI were calculated for each significant risk factor. RESULTS: Of the 10 376 newborns, 8 530 cases (82.21%) had 1-9 risk factors, and asphyxia occurred in 117 cases (1.13%) out of the 8 530 cases. In the 1 846 cases without risk factors, none had asphyxia (x2=25.6, p<0.01). The incidence of asphyxia increased with increasing numbers of risk factors, from 0.23% in newborns with one risk factor to 14.29% in newborns who had nine risk factors (r=0.96, p<0.01). Twelve significant risk factors identified were as follows: ominous fetal heart rate patterns (OR=17.1,95%CI:11.2-25.9), placenta abruption (OR=15.2, 95% CI: 4.5-51.8), maternal lung diseases (OR=11.5, 95% CI:1.4-91.3), fetal acidosis (OR=6.1, 95% CI:1.5-24.1), placenta previa (OR=5.0,95% CI:1.5-16.9), breech delivery (OR=4.5, 95% CI: 2.1-9.9), meconium stained amniotic fluid (OR=3.2, 95% CI:2.2-4.8), forcepsjassisted delivery (OR=3.2, 95%CI: 1.1-9.9), prolonged labor (OR=2.94, 95%CI:1.5-5.8), abnormal utero contraction (OR=2.8, 95% CI:1.7-4.6), and premature delivery (OR=2.5,95%CI:1.4-4.8). Cesarean section had a protective effect (OR=0.6, 95% CI:0.4-0.9) (all p<0.05). CONCLUSIONS: It is very important to prevent perinatal asphyxia by systematically examining prenatal risk factors and giving interventions for the newborns with risk factors, especially those with the above significant risk factors or with multiple risk factors. Proper cesareon section according to indications might be helpful to decrease the incidence of birth asphyxia.

[Clinical study on improving the diagnostic criteria for neonatal asphyxia].

OBJECTIVE: Diagnosing neonatal asphyxia solely according to Apgar score may lead to misdiagnosis. The aim of this study was to explore new and more accurate diagnostic criteria for neonatal asphyxia. METHODS: Totally 10 376 live born neonates in our hospital were consecutively enrolled into the study. The following five items related to birth asphyxia, i.e., antepartum high-risk factors, Apgar scores, umbilical artery blood pH, organ injury, differential diagnosis on the causes of low Apgar score cases were examined and registered. The relationship among the first 4 items were analyzed. By differential diagnosis, the sensitivity and specificity of each index on diagnosing asphyxia and their complementary value on each other were investigated. RESULTS: The items correlated well with each other (P < 0.01 or < 0.05) but were not entirely parallel and consistent; they could complement but could not substitute for each other. The sensitivity of antepartum high-risk factors, low Apgar scores, umbilical artery blood pH < 7.00 and organ injury was 100%, 100%, 44.44% and 100%, while the specificity was 17.99%, 98.90%, 96.05% and 96.62%, respectively. Of the 230 low Apgar score cases in this series only 50.9% coincided with asphyxia. For the 230 cases, when low Apgar score was combined with umbilical artery blood pH < 7.00, the sensitivity and specificity were 41% and 99.1% and when low Apgar score was combined with umbilical artery blood pH < 7.20, the sensitivity and specificity were 100% and 29.20%, respectively. After organ injury was added, the specificity was increased to 65.49%. When differential diagnosis was further added to exclude the other causes of low Apgar score cases, the misdiagnosis rate was minimized. CONCLUSION: Up to now, no single accurate index for diagnosing neonatal asphyxia is available. In order to increase diagnostic bases and reduce misdiagnosis, the criteria of sole Apgar score should be replaced by multi-index diagnostic criteria. Based on the present study, a set of integrated diagnostic criteria for neonatal asphyxia is proposed: (1) prenatal high-risk factors, (2) low Apgar scores (respiratory depression must present), (3) umbilical artery blood pH < 7.00, if only pH < 7.20, the items (2) (4) (5) must be present, (4) hypoxic-ischemic organ injury (at least one organ dysfunction), (5) the other causes of low Apgar scores should be excluded. The last 4 indexes should all be met and the first one serves as reference. If multi-organ (three or more organs) dysfunction and (or) hypoxic-ischemic encephalopathy are present, severe asphyxia can be diagnosed.