Navigating the microenvironment with flip and turn under quadrupole magnetophoretic steering control: Nanosphere- and nanorod-coated microbead.

The spatiotemporal accuracy of microscale magnetophoresis has improved significantly over the course of several decades of development. However, most of the studies so far were using magnetic microbead composed of nanosphere particle for magnetophoretic actuation purpose. Here, we developed an in-house method for magnetic sample analysis called quadrupole magnetic steering control (QMSC). QMSC was used to study the magnetophoretic behavior of polystyrene microbeads decorated with iron oxide nanospheres-coated polystyrene microbeads (IONSs-PS) and iron oxide nanorods-coated polystyrene microbeads (IONRs-PS) under the influence of a quadrupole low field gradient. During a 4-s QMSC experiment, the IONSs-PS and IONRs-PS were navigated to perform 180° flip and 90° turn formations, and their kinematic results (2 s before and 2 s after the flip/turn) were measured and compared. The results showed that the IONRs-PS suffered from significant kinematic disproportion, translating a highly uneven amount of kinetic energy from the same magnitude of magnetic control. Combining the kinematic analysis, transmission electron microscopy micrographs, and vibrating sample magnetometry measurements, it was found that the IONRs-PS experienced higher fluid drag force and had lower consistency than the IONSs-PS due to its extensive open fractal nanorod structure on the bead surface and uneven magnetization, which was attributed to its ferrimagnetic nature.

Efficient Catalytic Oxidation of 5-Hydroxymethylfurfural to 2,5-Furandicarboxylic Acid by Magnetic Laccase Catalyst.

2,5-Furandicarboxylic acid (FDCA) is a bio-based platform chemical for the production of polyethylene furanoate (PEF) and other valuable furanic chemicals. A magnetic laccase catalyst with (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) as the mediator has the remarkable capability of oxidizing 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA). Under optimal reaction conditions, a quantitative yield (90.2 %) of FDCA with complete HMF conversion was obtained after 96 h of reaction. More importantly, the magnetic laccase catalyst exhibited good recyclability and stability, maintaining 84.8 % of its original activity following six reuse cycles. This is the first report on the efficient catalytic oxidation of HMF to FDCA by using an immobilized enzyme catalyst.

Scale-up laccase production from Trametes versicolor stimulated by vanillic acid.

An efficient strategy for laccase production in Trametes versicolor cultures was developed using vanillic acid as the inducer. The optimized vanillic acid treatment strategy consisted of exposing 2-day-old mycelia cultures to 80 mg/L vanillic acid. After 4 days, laccase activity of 588.84 U/L was achieved in flasks which represented a 1.79-fold increase compared to the control. In 200-L airlift bioreactor, the maximal laccase activity reached up to 785.12 U/L using the optimized vanillic acid treatment strategy. The zymograms of culture supernatants revealed three bands with laccase activity, among which Lac1 and Lac2 were abundant laccase isoforms constitutively expressed, and Lac3 was an inducible isozyme by vanillic acid. The results of real-time quantitative PCR showed that the transcription level of lcc in T. versicolor cultures grown with vanillic acid for 7 days was about 5.64-fold greater than that without vanillic acid in flasks. In 200-L airlift bioreactor cultures of T. versicolor with addition of vanillic acid, the transcript level of lcc at day 7 was 2.62-fold higher than that in flasks with vanillic acid due to the good mass transfer and oxygen supply in the bioreactor system. This study provides a basis for understanding the induction mechanism of vanillic acid for laccase production and has good potential for industrial applications.

Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates.

Development of an efficient process intensification strategy for enhancing Pfu DNA polymerase production in recombinant Escherichia coli.

An efficient induction strategy that consisted of multiple additions of small doses of isopropyl-ß-D-thiogalactopyranoside (IPTG) in the early cell growth phase was developed for enhancing Pfu DNA polymerase production in Escherichia coli. In comparison to the most commonly used method of a single induction of 1 mM IPTG, the promising induction strategy resulted in an increase in the Pfu activity of 13.5% in shake flasks, while simultaneously decreasing the dose of IPTG by nearly half. An analysis of the intracellular IPTG concentrations indicated that the cells need to maintain an optimum intracellular IPTG concentration after 6 h for efficient Pfu DNA polymerase production. A significant increase in the Pfu DNA polymerase activity of 31.5% under the controlled dissolved oxygen concentration of 30% in a 5 L fermentor was achieved using the multiple IPTG induction strategy in comparison with the single IPTG induction. The induction strategy using multiple inputs of IPTG also avoided over accumulation of IPTG and reduced the cost of Pfu DNA polymerase production.

Chitosan multiple addition enhances laccase production from Trametes versicolor.

Chitosan multiple addition strategy was developed to improve laccase production from Trametes versicolor cultures. The optimized multiple addition strategy was carried out by two-time addition of 0.1 g L(-1) chitosan to a 2-day-old culture media, with 24-h interval between the treatments. Under these conditions, laccase activity of 644.9 U l(-1) was achieved on the seventh day and laccase production was improved by 93.5 % higher than the control. Chitosan treatment increased reactive oxygen species generation and extracellular protein concentration in the treated mycelia. In contrast, the inducer inhibited the mycelia growth. The result of the quantitative reverse transcription polymerase chain reaction showed that the copy number of the laccase gene transcript increased by 16.7-fold in the treated mycelia relative to the control. This study provides insight into some of the intrinsic metabolic processes involved in the upregulation of laccase production in the presence of chitosan inducer in fungal culture.

Immobilization of Trametes versicolor cultures for improving laccase production in bubble column reactor intensified by sonication.

The mycelia of Trametes versicolor immobilized in alginate beads provided higher laccase production than that in pelleted form. An efficient ultrasonic treatment enhanced laccase production from the immobilized T. versicolor cultures. The optimized treatment process consisted of exposing 36-h-old bead cultures to 7-min ultrasonic treatments twice with a 12-h interval using a fixed ultrasonic power and frequency (120 W, 40 kHz). Using the intensification strategy with sonication, laccase production increased by more than 2.1-fold greater than the untreated control in both flasks and bubble column reactors. The enhancement of laccase production by ultrasonic treatment is related to the improved mass transfer of nutrients and product between the liquid medium and the gel matrix. These results provide a basis for the large-scale and highly-efficient production of laccase using sonobioreactors.

Melatonin improves the survival of cryopreserved callus of Rhodiola crenulata.

An important aspect of the function of melatonin seems to be the mediation of stress caused by environmental and chemical factors. In the cryopreservation process, environmental changes including osmotic injury, desiccation, and low temperature can impose a series of stresses on plants. In this study, we evaluated the role of melatonin in stress protection during the process of cryopreservation using callus of an endangered plant species Rhodiola crenulata. The survival rate of the cryopreserved callus significantly increased when the callus was pretreated for 5 days with 0.1 µm melatonin prior to freezing in liquid nitrogen. Analysis of antioxidative activity following the pretreatment of callus with 0.1 µm melatonin showed a significant reduction in malondialdehyde production during various steps of cryopreservation. Enhanced peroxidase and catalase activity was observed in the callus after pretreatment with 0.1 µm melatonin compared to the control. These observations provide new evidence of the antioxidant/anti-stress function of melatonin, and it is the first report of its potential application in the preservation of elite endangered germplasm through the process of cryopreservation.

Efficient phenol degradation by laccase immobilized on functional magnetic nanoparticles in fixed bed reactor under high-gradient magnetic field.

Enzymatic degradation of emerging contaminants has gained great interest for the past few years. However, free enzyme often incurs high costs in practice. The immobilized laccase on the polyethylenimine (PEI)-functionalized magnetic nanoparticles (Fe3O4-NH2-PEI-laccase) was fabricated to efficiently degrade phenolic compounds continuously in a newly fixed bed reactor under a high-gradient magnetic field. The degradation rate of continuous treatment in the bed after 18 h was 2.38 times as high as that of batch treatment after six successive operations with the same treatment duration. Under the optimal conditions of volume fraction of nickel wires mesh, flow rate of phenol solution, phenol concentration, and Fe3O4-NH2-PEI-laccase amount, the degradation rate of phenol kept over 70.30% in 48 h continuous treatment. The fixed bed reactor filled with Fe3O4-NH2-PEI-laccase provided a promising avenue for the continuous biodegradation of phenolic compounds for industrial wastewater in practice.

Liver toxicity of macrolide antibiotics in zebrafish.

Macrolide antibiotics (macrolides) are among the most commonly prescribed antibiotics worldwide and are used for a wide range of infections, but macrolides also expose people to the risk of adverse events include hepatotoxicity. Here, we report the liver toxicity of macrolides with different structures in zebrafish. The absorption, distribution, metabolism, excretion and toxicology (ADMET) parameters of macrolide compounds were predicted and contrasted by utilizing in silico analysis. Fluorescence imaging and Oil Red O stain assays showed all the tested macrolide drugs induced liver degeneration, changed liver size and liver steatosis in larval zebrafish. Through RNA-seq analysis, we found seven co-regulated differentially expressed genes (co-DEGs) associated with metabolism, apoptosis and immune system biological processes, and two co-regulated significant pathways including amino sugar and nucleotide sugar metabolism and apoptosis signaling pathway. We found that only fosab of seven co-DEGs was in the two co-regulated significant pathways. fosab encoded proto-oncogene c-Fos, which was closely associated with liver diseases. The whole-mount in situ hybridization showed high transcription of c-Fos induced by macrolide compounds mainly in the liver region of zebrafish larvae. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) leakage assays revealed that macrolides exerts significant cytotoxic effects on L02 cells. qRT-PCR and western blot analysis demonstrated macrolides also promoted human c-Fos expression in L02 cells. The c-Fos overexpression significantly reduced cell viability by using CCK-8 assay. These data indicate that hepatotoxicity induced by macrolides may be correlated with c-Fos expression activated by these compounds. This study may provide a biomarker for the further investigations on the mechanism of hepatotoxicity induced by macrolide drugs with different structures, and extend our understanding for improving rational clinical application of macrolides.

Saussurea medusa cell suspension cultures for flavonoid production.

Saussurea medusa Maxim. is a valuable traditional Chinese herb. The flavonoids are the main active pharmaceutical compounds in this medicinal plant species and have effective anti-tumor and anti-inflammation properties. This species is now almost extinct in China because of over-exploitation. The establishment of plant cell cultures would be a promising alternative to avoid extinction of this species and establish cultivation for the production of bioactive flavonoids. The callus is induced from leaf explants of S. medusa on Murashiage and Skoog medium supplemented with 0.5 mg/L 6-BA, 2 mg/L NAA, 30 g/L sucrose, and 5 g/L agar. A fine cell suspension is established from the induced light-yellow calluses in the MS liquid medium with 30 g/L sucrose, 0.5 mg/L BA, and 2.0 mg/L NAA for biosynthesis of flavonoids. The kinetics of cell growth and flavonoid accumulation in the cell suspension cultures are investigated. The highest dry weight and flavonoid production reach 17.2 g/L and 607.8 mg/L respectively after 15 d. Significantly high antioxidant activity and flavonoids accumulate in the cell suspension cultures of S. medusa.

Effects of incident light intensity and light path length on cell growth and oil accumulation in Botryococcus braunii (Chlorophyta).

Botryococcus braunii was cultured in different light path length under different incident light intensity to investigate the effect of light on alga growth as well as hydrocarbon and fatty acid accumulation. Results indicated that longer light path length required higher incident light intensity in order to meet the light requirement of algal growth and hydrocarbon accumulation during the course of cultivation. However, hydrocarbon profile was only affected by the incident light intensity and not influenced by the light path length. High incident light intensity enhanced the accumulation of hydrocarbons with longer carbon chains. Besides, the fatty acid content and profiles were significantly influenced by both incident light intensity and light path. Higher fatty acid content and higher percentage of C18 and monounsaturated fatty acid components were achieved at the higher incident light intensity and lower light path length. Taken together, these results are benefit to improve its biomass and oil productivity through the optimization of light and photobioreactor design.

Separation of chlorogenic acid from honeysuckle crude extracts by macroporous resins.

Chlorogenic acid, one of the most bioactive compounds rich in the Chinese medicinal herb honeysuckle, is a natural antioxidant and serves as anti-inflammatory, anti-tumor, anti-mutagenic and anti-carcinogenic agent. An efficient preparative separation process of chlorogenic acid from honeysuckle crude extracts has been developed in the present study. HPD-850 resin offers the best adsorption capacity, and adsorption and desorption ratios for chlorogenic acid among the nine macroporous resins tested, and its adsorption rate at 25 degrees C fit best to the Langmuir isotherm. The adsorption capacity of HPD-850 resin was found to depend strongly on the pH value of the initial adsorption solution. The dynamic adsorption and desorption experiments have been carried out on a HPD-850 resin packed column to optimize the separation process of chlorogenic acid from honeysuckle crude extracts. After one run treatment with HPD-850 resin, the chlorogenic acid content in the final product was increased 4.46-fold from 11.2% to 50.0%, with a recovery yield of 87.9%. The preparative separation of chlorogenic acid can be easily and efficiently achieved via adsorption and desorption on HPD-850 resin, and the method developed will provide a potential approach for large-scale separation and purification of chlorogenic acid for its wide pharmaceutical use.

Farnesol-induced hyperbranched morphology with short hyphae and bulbous tips of Coriolus versicolor.

As the first fungal quorum sensing molecule, farnesol-induced morphological transition is usually studied in dimorphic fungi, but in basidiomycetes the morphological changes regulated by farnesol are rarely investigated. In this study, we found that farnesol made the basidiomycete Coriolus versicolor develop into a hyperbranched morphology with short hyphae and bulbous tips. Farnesol treatment resulted in a significant increase of intracellular oxidative stress level, which influenced the expression of several morphogenesis-related genes, and thereby led to the morphological changes. High oxidative stress level significantly stimulated the expression of laccase genes for improving intracellular laccase biosynthesis. The resulted hyperbranched morphology further accelerated the secretion of intracellular laccase into culture medium. As a result, extracellular laccase production reached a maximum of 2189.2 ± 54.7 U/L in farnesol-induced cultures, which was 6.8-fold greater than that of control cultures. SDS-PAGE and native-PAGE showed that farnesol increased laccase production by promoting the biosynthesis of three laccase isoforms. Together these results provide new opportunities in not only understanding the farnesol-regulated mycelial morphology in basidiomycetes, but also developing novel strategies for enhancing the production of secreted enzymes of biotechnological interest.

Separation of scutellarin from crude extracts of Erigeron breviscapus (vant.) Hand. Mazz. by macroporous resins.

Scutellarin, a flavone glycoside, popularly used in the treatment of heart disease, has been efficiently separated using macroporous resins from crude extracts of Chinese medicinal plant Erigeron breviscapus (vant.) Hand. Mazz. HPD-800 resin offered the best adsorption and desorption capacity for scutellarin among the eight macroporous resins tested, and its adsorption data at 25 degrees C fit best to the Langmuir isotherm. The dynamic adsorption and desorption experiments have been carried out on a HPD-800 resin packed column to optimize the separation process of scutellarin from the crude extracts of E. breviscapus. After one run treatment with HPD-800 resin, the scutellarin content in the product was increased 15.69-fold from 2.61% to 40.96% with a recovery yield of 95.01%. The preparative separation process via adsorption-desorption method developed in this study provides a new approach for scale-up separation and purification of scutellarin for its wide pharmaceutical use.

Quorum sensing molecule-farnesol increased the production and biological activities of extracellular polysaccharide from Trametes versicolor.

A novel strategy of exposing 2-day-old mycelia cultures to 0.8mM farnesol was developed to stimulate extracellular polysaccharide (EPS) production in Trametes versicolor submerged cultures. Farnesol, a quorum sensing molecule in fungi, could significantly increase EPS production by promoting polysaccharide biosynthesis and regulating mycelial morphology. EPS yield reached a maximum of 2.56g/L that was 2.7-fold greater than that of control cultures. Farnesol made T. versicolor develop into fluffy, loose and multi-hyphae morphology, which facilitated the excretion of intracellular polysaccharide into culture medium. Moreover, EPS from farnesol-induced cultures (EPS-F) with higher carbohydrate and uronic acid contents mainly contained high molecular weight polysaccharide (134kDa, 85%), and comprised glucose, mannose and galactose in a molar ratio of 34.2:2.1:1.0. These physicochemical properties led to stronger antioxidant and antitumor activities of EPS-F. This is the first report that farnesol can significantly improve the production of polysaccharide with higher biological activities. It provides a novel strategy to enhance the production and bioactivity of mushroom polysaccharide using microbial quorum sensing molecules.

Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor.

Effect of chitosan elicitor on growth and phenylethanoid glycosides (PeGs) accumulation in Cistanche deserticola cell suspension cultures was investigated. PeGs accumulation was dramatically improved by addition of selected chitosan at optimal elicitation conditions. Furthermore, a strategy of repeated addition of the chitosan elicitor for enhancing PeGs accumulation was developed. The chitosan elicitor of 10 mg l(-1)-medium repeatedly added on days 15 and 17 improved PeGs accumulation further, and the final PeGs production in the treated cell cultures of C. deserticola reached 364.6 mg l(-1), which was 3.4-fold higher than that of the control without elicitation. The increase of PeGs accumulation in C. deserticola cell suspension cultures was related to the increase of phenylalanine ammonium lyase activity stimulated by the chitosan elicitor.

Isolation, culture, and plant regeneration from Echinacea purpurea protoplasts.

A plant regeneration system from the isolated protoplasts of Echinacea purpurea L. using an alginate solid/liquid culture is described in the chapter. Viable protoplasts were isolated rom 100 mg of young leaves of 4-wk-old seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase, and 0.3 mol/L mannitol. After isolation and purification, the mesophyll protoplasts were embedded into 0.6% Na-alginate at the density 1 x 10(-5) mL and cultured in modified Murashige and Skoog (MS) culture medium supplemented with 0.3 mol/L sucrose, 2.5 micromol/L benzylaminopurine (BA), and 5.0 micromol/L 2,4-dichlorophenoxyacetic acid (2,4-D). The visible colonies were present after 4 wk of culture. The protoplast-derived clones were transferred onto gellan gum-solidified basal medium supplemented with 1.0 micromol/L BA and 2.0 micromol/L indole-3-butyric acid (IBA) and formed compact and green calli. Shoot development was achieved by subculturing the calli onto the same basal medium supplemented with 5.0 micromol/L BA and 2.0 micromol/L IBA. Further subculture onto basal medium resulted in the regeneration of complete plantlets.

Microwave-assisted extraction of solanesol from tobacco leaves.

Solanesol is the starting material for many high-value biochemicals, including co-enzyme Q10 and Vitamin K analogues. In the present study, a microwave-assisted extraction (MAE) technique has been developed for the fast extraction of solanesol from tobacco leaves. Compared to heat-reflux extraction, MAE reduced extraction time and obtained higher percentage extracted of solanesol. The effect of microwave on cell destruction of plant material was observed by scanning electron microscopy (SEM). The microwave-assisted extraction efficiency was further improved by adding NaOH into the extraction solvent, and the maximum percentage extracted of solanesol reached 0.91% (weight solanesol/weight tobacco) in 40 min at an optimum NaOH concentration of 0.05 M. The developed MAE integrated with saponification process provided an efficient method for solanesol recovery from tobacco leaf materials, and it also alleviated emulsification in the following separation and purification procedure as well.

Rapid determination of solanesol in tobacco by high-performance liquid chromatography with evaporative light scattering detection following microwave-assisted extraction.

Solanesol is the starting material for many high-value biochemicals, including coenzyme Q(10) and Vitamin-K analogues. The aim of the current study was to develop a reliable and fast analytical procedure for the determination of solanesol in tobacco using high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique. The HPLC conditions were Agilent C18 column using acetonitrile-isopropanol (60:40, v/v) as mobile phase at a flow rate of 1 ml/min. ELSD conditions were optimized at nebulizer-gas flow rate of 1.5 l/min and drift tube temperature of 65 degrees C. The method was validated to achieve the satisfactory precision and recovery, and the calibration range was 0.1-1.5 mg/ml. The developed analytical procedure was successfully applied to determine solanesol content in tobacco samples from different growing regions in China.