Design, Evaluation, and Implementation of Synthetic Isopentyldiol Pathways in Escherichia coli.

Isopentyldiol (IPDO) is an important raw material in the cosmetic industry. So far, IPDO is exclusively produced through chemical synthesis. Growing interest in natural personal care products has inspired the quest to develop a biobased process. We previously reported a biosynthetic route that produces IPDO via extending the leucine catabolism (route A), the efficiency of which, however, is not satisfactory. To address this issue, we computationally designed a novel non-natural IPDO synthesis pathway (route B) using RetroPath RL, the state-of-the-art tool for bioretrosynthesis based on artificial intelligence methods. We compared this new pathway with route A and two other intuitively designed routes for IPDO biosynthesis from various perspectives. Route B, which exhibits the highest thermodynamic driving force, least non-native reaction steps, and lowest energy requirements, appeared to hold the greatest potential for IPDO production. All three newly designed routes were then implemented in the Escherichia coli BL21(DE3) strain. Results show that the computationally designed route B can produce 2.2 mg/L IPDO from glucose but no IPDO production from routes C and D. These results highlight the importance and usefulness of in silico design and comprehensive evaluation of the potential efficiencies of candidate pathways in constructing novel non-natural pathways for the production of biochemicals.

Introduction of multilayered quantum dot nanobeads into competitive lateral flow assays for ultrasensitive and quantitative monitoring of pesticides in complex samples.

A competitive fluorescent lateral flow assay (CFLFA) is proposed for direct, ultrasensitive, quantitative detection of common pesticides imidacloprid (IMI) and carbendazim (CBZ) in complex food samples by using silica-core multilayered quantum dot nanobeads (SiO2-MQB) as liquid fluorescent tags. The SiO2-MQB nanostructure comprises a 200-nm SiO2 core and a shell of hundreds of carboxylated QDs (excitation/emission maxima ~365/631 nm), and can generate better stability, superior dispersibility, and higher luminescence than traditional fluorescent beads, greatly improving the sensitivity of current LFA methods for pesticides. Moreover, using liquid SiO2-MQB directly instead of via the conjugate pad both simplifies the structure of LFA system and improves the efficiency of immunobinding reactions between nanotags and the targets. Applying these methods, the established CFLFA realized the stable and accurate detection of IMI and CBZ in 12 min, with detection limits down to 1.94 and 14.79 pg/mL, respectively. The SiO2-MQB-CFLFA is practicable for application to real food samples (corn, apple, cucumber, and cabbage), and undoubtedly a promising and low-cost tool for on-site monitoring of trace pesticide residues.

Electrochemically mediated bioconversion and integrated purification greatly enhanced co-production of 1,3-propanediol and organic acids from glycerol in an industrial bioprocess.

In this study, we show how electrochemically mediated bioconversion can greatly increase the co-production of 1,3-propanediol and organic acids from glycerol in an industrial bioprocess using a Clostridum pasteurianum mutant. Remarkably, an enhanced butyrate formation was observed due to a weakened butanol pathway of the mutant. This allowed the strain to have a higher ATP generation for an enhanced growth, higher glycerol consumption and PDO production. The PDO titer reached as high as 120.67 g/L at a cathodic current of -400 mA, which is 33% higher than that without electricity, with a concurrent increase of butyric acid by 80%. To fully recover the increased PDO and organic acids, a novel downstream process combining thin film evaporation of PDO and esterification of organic acids with ethanol was developed. This enables the efficient co-production of PDO, ethyl acetate and ethyl butyrate with a high overall carbon use of 87%.

Strain evolution and novel downstream processing with integrated catalysis enable highly efficient coproduction of 1,3-propanediol and organic acid esters from crude glycerol.

Bioconversion of natural microorganisms generally results in a mixture of various compounds. Downstream processing (DSP) which only targets a single product often lacks economic competitiveness due to incomplete use of raw material and high cost of waste treatment for by-products. Here, we show with the efficient microbial conversion of crude glycerol by an artificially evolved strain and how a catalytic conversion strategy can improve the total products yield and process economy of the DSP. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72, and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The coproduction of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for the simultaneous production of three appealing products from the crude glycerol fermentation broth, which greatly improve the process economy and ecology.

A novel downstream process for highly pure 1,3-propanediol from an efficient fed-batch fermentation of raw glycerol by Clostridium pasteurianum.

An efficient downstream process without prior desalination was developed for recovering 1,3-propanediol (1,3-PDO) with high purity and yield from broth of a highly productive fed-batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3-PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3-PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3-PDO (< 80%). In order to improve the efficiency of final 1,3-PDO rectification, we examined alkaline hydrolysis to eliminate the ester impurities. By the use of 20% (w/w) water and 2% (w/w) sodium hydroxide, > 99% reduction of 1,3-PDO esters was achieved. This step conveniently provided free 1,3-PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3-PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3-PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high-salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3-PDO esters are crucial for the efficient separation of highly pure 1,3-PDO with acceptable yield from fermentation broth of raw glycerol.

Glabridin Liposome Ameliorating UVB-Induced Erythema and Lethery Skin by Suppressing Inflammatory Cytokine Production.

Glabridin, a compound of the flavonoid, has shown outstanding skin-whitening and anti-aging properties, but its water insolubility limits its wide application. Therefore, glabridin liposome (GL) has been developed to improve its poor bioavailability, while there are few studies to evaluate its amelioration of UVB- induced photoaging. This study is performed to investigate the amelioration of GL against UVB- induced cutaneous photoaging. The prepared GL has a spheroidal morphology with an average diameter of 200 nm. The GL shows lower cytotoxicity than glabridin, but it has a more effective role in inhibition of melanin. Moreover, the application of GL can effectively relieve UV radiation induced erythema and leathery skin, associated with the down-regulated expression of inflammatory cytokines (TNF-α, IL-6 and IL-10). Taken together, these results demonstrate that GL has potentials as topical therapeutic agents against UVB radiation induced skin damage through inhibiting inflammation.

Conformational adaptability determining antibody recognition to distomer: structure analysis of enantioselective antibody against chiral drug gatifloxacin.

Enantioselective antibodies have great potential to separate and detect chiral compounds. However, cross-reactivity of enantioselective antibodies to the distomer may limit the application. An in-depth understanding of interactions between antibodies and chiral drugs could be helpful to investigate antibody recognition to the distomer. In this study, a monoclonal antibody against chiral quinolone S-(-)-gatifloxacin (S-GAT) was produced and its Fab fragment was prepared by proteolysis. The S-GAT Fab exhibited 10% cross-reactivity against the R-enantiomer compared to that of the S-enantiomer in an indirect competitive enzyme-linked immunosorbent assay (icELISA). The crystal structures of the S-GAT Fab apo form and complex with S-GAT were analyzed, and molecular docking of the R-enantiomer was carried out. The ligand conformation was further studied using molecular dynamics simulations. The results showed that the distomer R-enantiomer could enter the chiral center recognition region of the antibody by adjusting the piperazine ring conformation. Meanwhile, the antibody binding cavity had obvious conformational adaptability during ligand binding. It demonstrated that conformational change of both ligand and antibody was the key reason why antibodies recognize the distomer. Restricting conformational adaptability could improve the enantioselective recognition ability of antibodies. This study provided a new explanation for the cross-reactivity of enantioselective antibodies to the distomer, and could help to modulate antibody enantioselectivity for immunoassay of chiral drugs.

Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition.

On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens.

Investigation of an Immunoassay with Broad Specificity to Quinolone Drugs by Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Data Sets and Advanced Quantitative Structure-Activity Relationship Analysis.

A polyclonal antibody against the quinolone drug pazufloxacin (PAZ) but with surprisingly broad specificity was raised to simultaneously detect 24 quinolones (QNs). The developed competitive indirect enzyme-linked immunosorbent assay (ciELISA) exhibited limits of detection (LODs) for the 24 QNs ranging from 0.45 to 15.16 ng/mL, below the maximum residue levels (MRLs). To better understand the obtained broad specificity, a genetic algorithm with linear assignment of hypermolecular alignment of data sets (GALAHAD) was used to generate the desired pharmacophore model and superimpose the QNs, and then advanced comparative molecular field analysis (CoMFA) and advanced comparative molecular similarity indices analysis (CoMSIA) models were employed to study the three-dimensional quantitative structure-activity relationship (3D QSAR) between QNs and the antibody. It was found that the QNs could interact with the antibody with different binding poses, and cross-reactivity was mainly positively correlated with the bulky substructure containing electronegative atom at the 7-position, while it was negatively associated with the large bulky substructure at the 1-position of QNs.

Molecular modeling application on hapten epitope prediction: an enantioselective immunoassay for ofloxacin optical isomers.

To deepen our understanding of the physiochemical principles that govern hapten-antibody recognition, ofloxacin enantiomers were chosen as a model for epitope prediction of small molecules. In this study, two monoclonal antibodies (mAbs) mAb-WR1 and mAb-MS1 were raised against R-ofloxacin and S-ofloxacin, respectively. The enantioselective mAbs have a high sensitivity and specificity, and the enantioselectivity is not affected by heterologous coating format reactions. The epitopes of the ofloxacin isomers were predicted using the hologram quantitative structure-activity relationship (HQSAR) and comparative molecular field analysis (CoMFA) approaches. The results consistently show that the epitope of the chiral hapten should be primarily composed of the oxazine ring and the piperazinyl ring and mAbs recognize the hapten from the side of this moiety. The enantioselectivity of mAbs is most likely due to the steric hindrance caused by the stereogenic center of the epitope. Modeling of chiral hapten-protein mimics reveals that ofloxacin isomers remain upright on the surface of the carrier protein. Suggestions to improve the enantioselectivity of antibodies against ofloxacin isomers were also proposed. This study provided a simple, efficient, and general method for predicting the epitopes of small molecules via molecular modeling. The epitope predictions for small molecules may create a theoretical guide for hapten design.

[Effect of dibutyl phthalate and di-(2-ethylhexyl) phthalate on urine SOD activity and MDA content in rats].

OBJECTIVE: To evaluate the effect of dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) on urine superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in rats. METHODS: According to 2×2 factorial analysis, 60 adult male SD rats were randomized into 10 groups (n=6), including a control group (fed with sesame oil), 3 DBP groups (fed with DBP at the doses of 30, 100 and 300 mg/kg), 3 DEHP groups (with DEHP at 50, 150, and 450 mg/kg), and 3 DBP+DEHP groups (with 30 mg/kg DBP+50 mg/kg DEHP, 100 mg/kg DBP+150 mg/kg DEHP, and 300 mg/kg DBP +450 mg/kg DEHP). The agents were administered in a single dose through gavage in a volume of 2 ml. After the treatments, the 24, 48, 72, and 96 h urine samples were collected to determine the SOD activity and MDA content. RESULTS: DBP and DEHP, either alone or in combination, significantly decreased SOD activity and increased MDA content in the urine collected at 24 h but not at the other time points. Such changes were gradually reversed with time. CONCLUSION: DBP or DEHP treatment alone can result in significant oxidative damage in the kidney of rats, and the toxic effect of the combined exposure is even more obvious.

[Adaptive network-based fuzzy inference system for the forecast of spectra].

Because of the function of independent adaptation and study of NN and the best nonlinear speculating ability of fuzzy system, the adaptive network-based fuzzy inference system (ANFIS) which is composed of them absorbs their virtue. When applying it to the data analysis and model construction, the authors got the good nonlinear forecast by learning data, such as fluorescence spectrum. Fluorescence spectrometry is an important means of researching inside structure of molecule, which works easy, and features rapidness and high precision. So its forecast is more important. In the present paper, the authors utilize the spectra of the N2 to calculate, and prove that the means can forecast important spectrum information, the error is very small, less than 1. 66 percent, which is conformed to meet the demand of the experiment. In short, the approach is workable.