Highly Efficient Loading of Procaine on Water-Soluble Carbon Dots toward Long-Acting Anesthesia.

Safe and efficient local anesthetic delivery carriers are crucial for long-term anesthesia and analgesics in clinical treatment. But currently, most of the local anesthetic carriers still have some disadvantages such as low drug-loading capacity, drug leakage, and potential side effects. Here, we report red-emissive carbon dots (Cys-CDs) synthesized by choosing cysteine and citric acid as precursors, which contain a large and intact sp2-domain with rich hydrophilic groups around the edge. The special structure of Cys-CDs is conducive to the efficient loading of procaine (PrC) via strong π-π stacking interactions. Based on the strong noncovalent interactions between them, the PrC loaded on Cys-CDs achieved slow release in vitro and had a long-lasting nerve blocking effect in vivo, which is 4-fold more than that of free PrC. More importantly, PrC/Cys-CDs do not cause any toxicity and inflammation during treatment owing to slow release of PrC and good water solubility of Cys-CDs, thus demonstrating the potential clinical application of CDs in long-lasting analgesia.

Enhanced Dissolution of 7-ADCA in the Presence of PGME for Enzymatic Synthesis of Cephalexin.

Enzymatic catalysis has been recognized as a green alternative to classical chemical route for synthesis of cephalexin (CEX). However, its industrial practice has been severely limited by the low productivity due to the low solubility of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and high hydrolysis of D-phenylglycine methyl ester (PGME). In this work, the enhanced dissolution of 7-ADCA in the presence of PGME for efficient enzymatic synthesis of CEX was investigated. Results showed that the solubility of 7-ADCA in water could be improved by PGME. Moreover, supersaturated solution of 7-ADCA could be created in the presence of PGME by a pH shift strategy. The supersaturated solution of 7-ADCA possess good stability, which could be explained in terms of the inhibition of 7-ADCA precipitation due to the presence of PGME. The interaction between 7-ADCA and PGME is explored by spectroscopic determination and DFT analysis and the mechanism of enhanced dissolution of 7-ADCA in the presence of PGME is discussed and proposed. The feasibility of supersaturated solution of 7-ADCA for the enzymatic synthesis of CEX is evaluated. It was demonstrated that high conversion ratio (> 95.0%) and productivity (> 240.0 mmol/L/h) was obtained under a wide range of reaction conditions, indicating that the supersaturated solution system was highly superior to conventional homogeneous solution system. The information obtained in this work will be helpful to industrial production of CEX via enzymatic route.

Efficient enzymatic synthesis of cephalexin in suspension aqueous solution system.

An efficient method for the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and d-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase (IPGA) as catalyst in a suspension aqueous solution system was developed, where the reactant 7-ADCA and product CEX are mainly present as solid particles. The effects of key factors on the enzymatic synthesis were investigated. Results showed that continuous feeding of PGME was more efficient for the synthesis of CEX than the batch mode. Under the optimized conditions, the maximum 7-ADCA conversion ratio of 99.3% and productivity of 200 mmol/L/H were achieved, both of which are much superior to the homogeneous aqueous solution system. Besides, IPGA still retained 95.4% of its initial activity after 10 cycles of enzymatic synthesis, indicating the excellent stability of this approach. The developed approach shows great potential for the industrial production of CEX via an enzyme-based route.

FAM83D knockdown regulates proliferation, migration and invasion of colorectal cancer through inhibiting FBXW7/Notch-1 signalling pathway.

Colorectal cancer is one of the most common malignant tumors in the digestive system, and in China its incidence is rising in recent years. The FAM83D family with sequence similarity 83, member D is associated with the occurrence and development of various cancers. However, in human colorectal cancer, the biological function of FAM83D and its molecular mechanism are still little known. In our study, we found that FAM83D mRNA expression level was markedly up-regulated in colorectal cancerous tissues when compared with that of adjacent normal colon tissues. We also found that the protein and mRNA expression levels of FAM83D are dramatically increased in human colorectal cancer cell lines, including Caco-2, RKO, DLD-1, HT-29, LoVo, SW480, and HCT116, especially in SW480 and HCT116 cells, when compared with that of human normal colon epithelial cell line (NCM460). Next, in HCT116 and SW480 cells, the biological function of FAM83D was examined. FAM83D-knockdown notably inhibited cell proliferation and colony formation. Cell apoptosis was promoted by FAM83D knockdown. In addition, FAM83D siRNA decreased cell migration and invasion. Moreover, FAM83D knockdown up-regulated the protein expression level of F-box and WD repeat domain-containing 7 (FBXW7), but diminished the Notch1 protein expression level. It also found that FBXW7 siRNA reverses the suppressive effect of FAM83D knockdown on Notch1 protein expression. Notch1 overexpression reversed the effect of FAM83D knockdown on colorectal cancer cell proliferation, cell migration and invasion. In conclusion, FAM83D knockdown promoted colorectal cancer cell apoptosis, inhibited cell proliferation, cell migration and invasion, which might be associated with inhibiting the FBXW7/Notch1 signal pathway. Our findings indicated that FAM83D is a promising molecular target for colorectal cancer treatment.

Effect of nitrogen, carbon sources and agitation speed on acetoin production of Bacillus subtilis SF4-3

Background: Currently, microbial fermentation method has become the research hotspot for acetoin production. In our previous work, an acetoin-producing strain, Bacillus subtilis SF4-3, was isolated from Japanese traditional fermented food natto. However, its conversion of glucose to acetoin was relatively low. In order to achieve a high-efficient accumulation of acetoin in B. subtilis SF4-3, main medium components and fermentation conditions were evaluated in this work. Results: The by-products analysis showed that there existed reversible transformation between acetoin and 2,3-butanediol that was strictly responsible for acetoin production in B. subtilis SF4-3. The carbon sources, nitrogen sources and agitation speed were determined to play crucial role in the acetoin production. The optimal media (glucose-H2O 150 g/L, yeast extract 10 g/L, corn steep dry 5 g/L, urea 2 g/L, K2HPO4 0.5 g/L, MgSO4 0.5 g/L) were obtained. Furthermore, the low agitation speed of 300 r/min was found to be beneficial to the reversible transformation of 2,3-butanediol for acetoin production in B. subtilis SF4-3. Eventually, 48.9 g/L of acetoin and 5.5 g/L of 2,3-butanediol were obtained in a 5-L fermenter, and the specific production of acetoin was 39.12% (g/g), which accounted for 79.90% of the theoretical conversion. Conclusions: The results indicated acetoin production of B. subtilis SF4-3 was closely related to the medium components and dissolved oxygen concentrations. It also provided a method for acetoin production via the reversible transformation of acetoin and 2,3-butanediol.

Optimization of fermentation medium for acetoin production by Bacillus subtilis SF4-3 using statistical methods.

To improve the acetoin-producing ability of Bacillus subtilis SF4-3, isolated from "natto," a Japanese traditional food, the fermentation medium was optimized in shake-flask fermentation by statistically designed methods. Based on results of the single-factor experiment, orthogonal experiment, and Plackett-Burman design, yeast extract, corn steep liquor, and urea were identified as showing significant influence on the acetoin production. Subsequently, the optimum combination of the three factors was investigated by the Box-Behnken design (BBD) of response surface methodology (RSM) in order to further enhance the acetoin production. The maximum acetoin yield of 45.4 g/L was predicted when the concentrations of yeast extract, corn steep liquor, and urea were 8.5 g/L, 14.6 g/L, and 3.8 g/L, respectively. The results were further confirmed in triplicate experiments using the optimized medium (glucose 160 g/L, yeast extract 8.5 g/L, corn steep liquor 14.6 g/L, urea 3.8 g/L, manganese sulfate 0.05 g/L, ferrous sulfate 0.05 g/L), and an acetoin yield of 46.2 g/L was obtained in the validation experiment, which was in agreement with the prediction. After the optimization of medium components, an increase of 36.28% in acetoin production was achieved in comparison to that at the initial medium levels.

Isolation of acetoin-producing Bacillus strains from Japanese traditional food-natto.

In this study, Bacillus strains with an ability to produce acetoin were isolated from a Japanese traditional food, natto, on the basis of the Voges-Proskauer (VP) reaction, and strain SF4-3 was shown to be a predominant strain in acetoin production. Based on a variety of morphological, physiological, and biochemical characteristics as well as the nucleotide sequence analysis of 16S rDNA, the strain SF4-3 was identified as Bacillus subtilis. When it was incubated at 37°C with a speed of 180 rpm for 96 hr in the flasks, the maximum acetoin concentration was up to 33.90 g/L. The fermentation broths were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses; the results showed that the major metabolite was acetoin, and the purity could reach more than 95% without butanedione and 2,3-butanediol, which were usually produced together with acetoin in other strains. A novel aqueous two-phase system (ATPS) composed of hydrophilic solvents and inorganic salts was developed for the extraction of acetoin from fermentation broths. The ethanol and dipotassium hydrogen phosphate system could be used to extract acetoin from fermentation broths. The influences of phase composition on partition of acetoin were investigated. The maximum partition coefficient (9.68) and recovery (94.6%) of acetoin were obtained, when 25% (w/w) dipotassium hydrogen phosphate and 24% (w/w) ethanol were used.