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.

[Variety and standard arrangement of Yao medicine in Euphorbiaceae].

The names, basic sources, medicinal parts, efficacy and standards of the medicinal materials in Euphorbiaceae were systematically collated and analyzed by the textual research for Yao medicine monographs in this paper. The results showed that there were great differences in the names, basic sources, medicinal parts and efficacy of some medicinal materials recorded in different literatures. There were 19 genera and 60 species(including varieties) of Euphorbiaceae of Yao medicine, involving 50 kinds of medicinal materials. Among them, there were 42 kinds of single basic sources medicine, 8 kinds of multi basic sources medicine, 28 kinds of root medicine, 26 kinds of whole plant medicine, 25 kinds of unique Yao medicine, accounting for 50%, 11 kinds of cross with Chinese medicinal materials, accounting for 22%. There were 21 kinds of Yao medicine standards at all levels, but only 1 kind of Laoban medicine and 2 kinds of Yao medicine standards. The Yao medicine in Euphorbiaceae could be named by means of transliteration of Yao language/Yao language transliteration/Chinese medicine name, Laoban medicine, plant morphology, medicinal properties, color and smell of medicine, while the medicinal parts and efficacy of the same medicinal name were different from those of traditional Chinese medicine. Therefore, the name and basic sources of the medicinal materials in Euphorbiaceae were not standardized, and the quality standard is not perfect. The above results provided a reference for the construction and improvement of quality standard system, the promotion of the production of medicinal materials and clinical medication standards, and the further development and utilization of Euphorbiaceae.

Double C-N bond cleavages of N-alkyl 4-oxopiperidinium salts: access to unsymmetrical tertiary sulfonamides.

In this paper, regiospecific, double intraannular C-N bond cleavages of N-alkyl 4-oxopiperidinium salts are presented. The reaction sequence involves a charge-transfer complex, in situ formed between sulfonyl chloride and N-methylmorpholine, which induces S-Cl bond homolysis of sulfonyl chloride, yielding a reactive sulfonyl radical that further induces the double C-N bond cleavages of N-alkyl 4-oxopiperidinium salt. The secondary amine thus produced was trapped by sulfonyl chloride to yield the desired sulfonamide product. The key feature of this protocol is that two intraannular C-N bonds of the 4-oxopiperidine ring are cleaved in one step under metal- and oxidant-free conditions.

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.

Effects of Lyrm1 knockdown on mitochondrial function in 3 T3-L1 murine adipocytes.

To explore the effects of Lyrm1 knockdown on the mitochondrial function of 3 T3-L1 adipocytes using small interfering RNA (siRNA). 3 T3-L1 preadipocytes were infected with either a negative control (NC) expression lentivirus or a Lyrm1-shRNA expression lentivirus and induced to differentiate. The knockdown efficiency of Lrym1-specific shRNA in 3 T3-L1 cells was evaluated by real-time PCR. The ultrastructure of the mitochondria in adipocytes was visualized using transmission electron microscopy after differentiation. The levels of mitochondrial DNA copy numbers and Ucp2 mRNA were detected by real-time quantitative PCR. The levels of ATP production was detected using a photon-counting luminometer. The mitochondrial membrane potential and ROS levels of cells were analyzed with a FACScan flow cytometer using Cell Quest software. Cells transfected with lentiviral-Lyrm1-shRNA showed a significantly reduced transcription of Lyrm1 mRNA compared with NC cells. The size and ultrastructure of mitochondria in Lyrm1 knockdown adipocytes was similar to those of the NC cells. There was no significant difference in mtDNA copy number between the two groups. The total level of ATP production, mitochondrial membrane potential and Ucp2 mRNA expression levels were dramatically increased in adipocytes transfected with Lyrm1 RNAi. Furthermore, the level of ROS was dramatically decreased in Lyrm1 knockdown adipocytes. Knockdown of the Lyrm1 gene in adipocytes resulted in dramatically increased cellular ATP production, mitochondrial membrane potentials and levels Ucp2 mRNA, while ROS levels were significantly decreased. These results imply that mitochondrial function is improved in adipocytes after the knockdown of Lyrm1.

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway.

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.

Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early retinal development in zebrafish, Danio rerio. Zebrafish embryos were immediately exposed to different concentrations (0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg) of PCBs per liter of medium at 28.5 °C. Embryos were assessed at 30, 48, 72 and 96 h post-fertilization (hpf) for changes in embryonic survival rate, development, larval retinal morphology and ultrastructure of the retina. The results show that PCB exposure decreased the survival rate of embryos in a time- and dose-dependent manner. Embryos exposed to the higher concentrations of PCBs (0.5, 1.0 and 2.0 mg l(-1) ) displayed obvious gross morphological deformities. At 72 hpf, the retinal layer development of zebrafish was delayed at higher PCB concentrations (1.0 mg l(-1) ). At 96 hpf, irregularity of photoreceptor cells arrangement and thickening of photoreceptor and ganglionic layers were observed in PCB-treated larvae at concentrations of 0.25-1 mg l(-1) . Ultrastructural examination showed signs of growth inhibition of the photoreceptor outer segment at 0.25-1 mg l(-1) PCB exposure at 72 hpf, as well as the appearance of massive vacuoles and holes inside the outer segments in the PCB exposure group at 96 hpf. These results suggest that embryonic exposure to moderate and high levels of PCBs induced developmental deficits in zebrafish retinas, particularly in photoreceptor cells.

UCP4 overexpression improves fatty acid oxidation and insulin sensitivity in L6 myocytes.

Obesity, which is caused by energy uptake being greater than energy expenditure, is widely prevalent today. Currently, only a limited number of efficient interventional strategies are available for the prevention of obesity. Previous studies have shown that UCP4 transcription occurs at a considerable level in mouse skeletal muscle; however, the exact functions of UCP4 remain unclear. In this study, we investigated the effect of UCP4 on mitochondrial function and insulin sensitivity in mature L6 myocytes. UCP4 overexpression in L6 myocytes induced increased mitochondrial carnitine palmitoyltransferase 1A (CPT1A) and decreased citrate synthase (CS) mRNA in the basal condition (i.e., in the absence of insulin). UCP4 overexpression significantly improved insulin sensitivity, increased tyrosine phosphorylation of IRS-1 in the presence of insulin, and significantly reduced intracellular triglyceride (TG). Additionally, intracellular ATP content and mitochondrial membrane potential were downregulated. We also observed that intracellular ROS, mitochondrial morphology, and mitochondrial mtDNA copy number were maintained upon UCP4 expression, with no change in mitochondrial fusion and fission. In summary, our findings provide evidence to show that UCP4 overexpression reduced the insulin sensitivity and mitochondrial fatty acid oxidation of L6 myocytes. These findings support the notion that UCPs are ideal targets for treatment of insulin resistance.

Monoclonal antibody to six transmembrane epithelial antigen of prostate-4 influences insulin sensitivity by attenuating phosphorylation of P13K (P85) and Akt: possible mitochondrial mechanism.

We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002 mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.

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.

Overexpression of LYRM1 induces mitochondrial impairment in 3T3-L1 adipocytes.

Homo sapiens LYR motif containing 1 (LYRM1) is a recently discovered gene involved in adipose tissue homeostasis and obesity-associated insulin resistance. The exact mechanism by which LYRM1 induces insulin resistance has not yet been fully elucidated. In this study, we demonstrated that the overexpression of LYRM1 in 3T3-L1 adipocytes resulted in reduced insulin-stimulated glucose uptake, an abnormal mitochondrial morphology, and a decrease in intracellular ATP synthesis and mitochondrial membrane potential. In addition, LYRM1 overexpression led to excessive production of intracellular of reactive oxygen species. Collectively, our results indicated that the overexpression of LYRM1 caused mitochondrial dysfunction in adipocytes, which might be responsible for the development of LYRM1-induced insulin resistance.

Short report: Tissue-specific expression profiles of the uncoupling protein family in normal control mice and genetically ob/ob mice.

Uncoupling proteins (UCPs) located in the inner mitochondrial membrane are involved in the regulation of energy balance. Thus far, 5 UCP isoforms have been identified, but controversies exist in the research focused on the function of the UCPs (except UCP1) in the pathogenesis of obesity. Because of the known cross-reactivity of the antibodies presently available for the detection of UCP proteins, this study systematically analyzed the differential tissue expression profiles of the 5 UCP isoforms in lean control mice and ob/ob mice by using real-time polymerase chain reaction (PCR) analysis. The results show that the tissue-specific expression patterns of individual isoforms in normal and ob/ob mice are considerably different; this will provide new insights into the functions of UCPs in the pathogenesis of genetic obesity.

Gene expression profiles of adipose tissue of high-fat diet-induced obese rats by cDNA microarrays.

To better understand the molecular basis of dietary obesity, we examined adipose tissue genes differentially expressed in a well-characterized rat model of high-fat diet (HFD)-induced obesity using cDNA microarrays. Male Sprague-Dawley rats were fed either the HFD or the normal diet. Seven weeks later, the weights of obese models (362.92 ± 39.65 g) were significantly higher than those of normal control rats (315.22 ± 42.30 g, P < 0.01) and the wet weights of adipose tissue of rats fed with HFD (9.29 ± 5.14 g) were significantly higher than those of normal control rats (4.09 ± 2.69 g, P < 0.01), which confirmed the successful preparation of obese models. cDNA microarrays containing 9 216 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 532, 154, and 22 genes were differently expressed over 2-, 3-, and 5-fold, respectively. The analysis of gene expression profiles indicated that 276 genes were up-regulated and 432 genes were down-regulated in response to HFD-induced obesity. Different clusters of genes associated with lipid metabolism, extracellular matrix, signal transduction, cytoskeleton, cell apoptosis, etc., such as VLCS-H2, DGAT, ACADVL, PHYH, SCD, ACACA, ACS, MMP-2, MMP-15, CD38, CAMK2D, CACNA1F, CAPZA2, TMOD3, ARPC2, KNS2, TPM1, MAPK8, GADD45B, DAXX, TOK-1, PRKACA, STAT6, were concerned.

The lin-4 gene controls fat accumulation and longevity in Caenorhabditis elegans.

Previous studies have determined that lin-4, which was the first miRNA to be discovered, controls the timing of cell fate determination and life span in Caenorhabditis elegans. However, the mechanism of lin-4 involvement in these processes remains poorly understood. Fat storage is an essential aspect of the life cycle of organisms, and the function of lin-4 in fat accumulation is not clear. In this study, we showed that the fat content is reduced remarkably in C. elegans lin-4 mutants. Quantitative RT-PCR analysis revealed a considerable decrease in the levels of SBP-1 and OGA-1 mRNA in lin-4 mutants. We also showed that lin-4 mutants have a significantly shorter life span than wild-type worms. DCF assay experiments showed that the reactive oxygen species (ROS) levels increased and mitochondrial DNA (mtDNA) copy number decreased in loss-of-function lin-4 mutants. These mutants also showed attenuation of locomotion. Taken together, our findings suggest that lin-4 may play an important role in regulating fat accumulation and locomotion and that lin-4 may control the life span of C. elegans by mediating ROS production.