Molecular mechanism of a novel root-end filling material containing zirconium oxide on the osteogenic/odontogenic differentiation of human osteosarcoma MG-63 cells.

Although the novel root-end filling material containing zirconium oxide (NRFM-Zr) which is hydroxyapatite-based may promote osteoblast differentiation, the molecular mechanism remains unclear. The aim of this study is to investigate it underlying the osteogenic/odontogenic differentiation of human osteosarcoma MG-63 cells induced by NRFM-Zr, compared with calcium silicate-based mineral trioxide aggregate (MTA), and glass ionomer cement (GIC). Firstly, three different types of root filling materials were co-cultured with MG-63 cells, and their cell toxicity, alkaline phosphatase (ALP) activity, and calcium ion concentration were evaluated. Next, gene expression profiling microarray was employed to analyze the impact of the materials on the gene expression profile of MG-63 cells. The results of cell viability revealed that NRFM-Zr group had no significant difference compared to the negative control group. After 5 and 7 days of cultivation, both the NRFM-Zr and MTA groups exhibited significantly higher ALP activity compared to the negative control (p < 0.05). Moreover, the NRFM-Zr group had the highest calcium ion concentration, while the GIC group was the lowest (p < 0.05). Gene expression profiling microarray analysis identified 2915 (NRFM-Zr), 2254 (MTA) and 392 (GIC) differentially expressed genes, respectively. GO functional and KEGG pathway analysis revealed that differentially expressed genes of NRFM-Zr, MTA and GIC participated in 8, 6 and 0 differentiation-related pathways, respectively. Comparing the molecular mechanisms of osteogenic/odontogenic differentiation induced by hydroxyapatite-based NRFM-Zr and calcium silicate-based MTA, it was found that they shared similarities in their molecular mechanisms of promoting osteogenic differentiation. NRFM-Zr primarily promotes differentiation and inhibits cell apoptosis, thereby enhancing osteogenic/odontogenic differentiation of MG-63 cells. Furthermore, the inducing efficacy of NRFM-Zr was found to be superior to MTA.

La-Doped Ultrahigh-Nickel Layered Oxide Cathode with Enhanced Cycle Stability for Li-Ion Batteries.

Currently, ultrahigh-nickel layered oxide is one of the most promising cathodes for lithium-ion batteries, with the advantages of high theoretical capacity and low cost. However, some problems in ultrahigh-nickel layered oxides are more serious, such as irreversible structural transformation, particle cracking, and side reactions at the electrode/electrolyte interface, resulting in the fast decay of the discharge capacity and midpoint potential. In this work, La doping is introduced into ultrahigh-nickel layered LiNi0.9Co0.1O2 oxide to improve the cycle stability on both discharge capacity and midpoint potential. As demonstrated, La can be doped successfully into the subsurface of LiNi0.9Co0.1O2 oxide, and the morphology of the oxide microspheres is not changed obviously by La doping. Compared with the pristine sample, the La-doped sample presents improved electrochemical performance, especially good cycle stabilization on both discharge capacity and midpoint potential. In addition, after a long-term cycle, the La-doped sample still maintains a relatively complete spherical morphology. It means that the pillaring effect of La with a large radius is helpful in accommodating the volume change caused by the insertion/extraction of Li ions, thus easing the anisotropic stress accumulation and microcrack growth inside the microspheres of the La-doped sample.

[Effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in rats with osteoporosis and periodontitis].

PURPOSE: To investigate the effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in mice with osteoporosis and periodontitis. METHODS: Thirty rats were randomly divided into 3 groups, with 10 rats in each group. They were divided into control group, ovariectomized periodontitis group and chitosan oligosaccharide treatment group. Except for the control group, the other two groups were ovariectomized and smeared with Porphyromonas gingivalis fluid to establish the model of osteoporosis with periodontitis. Four weeks after ligation, the rats in chitosan oligosaccharide treatment group were gavaged with 200 mg/kg chitosan oligosaccharide, and the other two groups were gavaged with equal volume of normal saline once a day for 90 days. The periodontal tissues of each group were observed before administration, and the bone mineral density of rats was detected by dual energy X-ray animal bone mineral density and body composition analysis system. After 90 days of administration, the bone mineral density was detected again. After administration, blood was collected from tail vein, and the contents of serum alkaline phosphatase (ALP), bone Gla protein (BGP) and tartrate resistant acid phosphatase 5b (TRACP5b) were measured by enzyme-linked immunodeficient assay. The gingival index and periodontal attachment loss of rats in each group were obtained by visual examination and exploratory examination. The maxilla was removed, and the distance from the enamel cementum boundary to the alveolar crest was measured to obtain alveolar bone absorption value. H-E staining was used to observe the pathology of maxilla in each group. RT-PCR and Western blot were used to detect the nuclear factors in periodontal tissue of rats in each group. SPSS 22.0 software package was used for statistical analysis. RESULTS: Before administration, the gums of the control group were pink without bleeding, and the gums of the other two groups were red and swollen with slight bleeding. After administration, compared with the control group, the bone mineral density, serum ALP, BGP of ovariectomized periodontitis group decreased significantly(P＜0.05); while TRACP5b, gingival index, loss of periodontal attachment and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue increased significantly(P＜0.05). Compared with the ovariectomized periodontitis group, the bone mineral density, serum ALP, BGP were significantly increased(P＜0.05); while TRACP5b, gingival index, periodontal attachment loss and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue were significantly decreased (P＜0.05). In the ovariectomized periodontitis group, the periodontal tissue combined with epithelium was separated from the tooth surface, the dental pocket was obvious and deep, and the height of alveolar bone decreased. Although dental pocket could be observed in the periodontal tissue of rats treated with chitosan oligosaccharide, it was not obvious, and new bone appeared around the alveolar bone. CONCLUSIONS: Chitosan oligosaccharide can induce biochemical indexes of bone metabolism to become normal, alleviate the symptoms of periodontitis, this may be related to the inhibition of IKK/NF-κB pathway by chitosan oligosaccharide.

A Fully Implantable and Programmable Epidural Spinal Cord Stimulation System for Rats with Spinal Cord Injury.

Epidural spinal cord stimulation (ESCS) is a potential treatment for the recovery of the motor function in spinal cord injury (SCI) patients. Since the mechanism of ESCS remains unclear, it is necessary to study the neurophysiological principles in animal experiments and standardize the clinical treatment. In this paper, an ESCS system is proposed for animal experimental study. The proposed system provides a fully implantable and programmable stimulating system for complete SCI rat model, along with a wireless charging power solution. The system is composed of an implantable pulse generator (IPG), a stimulating electrode, an external charging module and an Android application (APP) via a smartphone. The IPG has an area of 25×25 mm2 and can output 8 channels of stimulating currents. Stimulating parameters, including amplitude, frequency, pulse width and sequence, can be programmed through the APP. The IPG was encapsulated with a zirconia ceramic shell and two-month implantable experiments were carried out in 5 rats with SCI. The main focus of the animal experiment was to show that the ESCS system could work stably in SCI rats. The IPG implanted in vivo can be charged through the external charging module in vitro without anesthetizing the rats. The stimulating electrode was implanted according to the distribution of ESCS motor function regions of rats and fixed on the vertebrae. The lower limb muscles of SCI rats can be activated effectively. The two-month SCI rats needed greater stimulating current intensity than the one-month SCI rats The results indicated that the stimulating system provides an effective and simplified tool for studying the ESCS application in motor function recovery for untethered animals.

Enhanced production of poly-γ-glutamic acid via optimizing the expression cassette of Vitreoscilla hemoglobin in Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is a natural polymer with various applications, and its high-viscosity hinders oxygen transmission and improvement of synthesis level. Vitreoscilla hemoglobin (VHB) has been introduced into various hosts as oxygen carrier, however, its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis. Here, we want to optimize the expression cassette of VHB for γ-PGA production. Firstly, our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation (Tat) signal peptides (SPYwbN, SPPhoD and SPTorA) into VHB expression cassette, and the best performance was attained by SPYwbN from Bacillus subtilis, the γ-PGA yield of which was 18.53% higher than that of control strain, and intracellular ATP content and oxygen transfer coefficient (KLa) were increased by 29.71% and 73.12%, respectively, indicating that VHB mediated by SPYwbN benefited oxygen transfer and ATP generation for γ-PGA synthesis. Furthermore, four promoters were screened, and P vgb was proven as the more suitable promoter for VHB expression and γ-PGA synthesis, and γ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%. Finally, WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation, and 46.39 g/L γ-PGA was attained by glucose feeding, increased by 49.26% compared with the initial yield (31.01 g/L). Taken together, this study has attained an efficient VHB expression cassette for oxygen transfer and γ-PGA synthesis, which could also be applied in the production of other metabolites.

Rational engineering of cofactor specificity of glutamate dehydrogenase for poly-γ-glutamic acid synthesis in Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis.

Disordered Metabolism and Repair Mechanism: Mitochondria Influenced by Cationic and Neutral Nanoparticles.

Limited understanding of mitochondria disorders that induced by nanoparticles is a stumbling block for anti-cancer drug delivery targeting strategy. In present study, C6 glioma cells were exposed to aminated and alkylated SiO2 nanoparticles for mitochondrion disordering and cell metabolism study. Collective results showed that aminated nanoparticles tend to trigger the cell-repair mechanism in cancer cells while alkylated nanoparticles could cause irreversible damages on cancer cells, although both types of the particles were proved to damage mitochondrion. The underlying mechanism show that aminated nanoparticles induced proton-stuck effect in mitochondrion and self-repairing in cancer cells by up-regulating p21. Otherwise, alkylated nanoparticles damaged mitochondrion and induced phosphorylated cyclin E accumulation lead to Fbw7 down-regulation caused further S phase arrest and severe late apoptosis. This work can help us elucidate the mechanism of the clinic application of nano-drug carriers.

Enhanced production of heterologous proteins via engineering the cell surface of Bacillus licheniformis.

Cell surface engineering was proven as the efficient strategy for enhanced production of target metabolites. In this study, we want to improve the yield of target protein by engineering cell surface in Bacillus licheniformis. First, our results confirmed that deletions of D-alanyl-lipoteichoic acid synthetase gene dltD, cardiolipin synthase gene clsA and CDP-diacylglycerol-serine O-phosphatidyltransferase gene pssA were not conducive to cell growth, and the biomass of gene deletion strains were, respectively, decreased by 10.54 ± 1.43%, 14.17 ± 1.51%, and 17.55 ± 1.28%, while the concentrations of total extracellular proteins were improved, due to the increases of cell surface net negative charge and cell membrane permeability. In addition, the activities of target proteins, nattokinase, and α-amylase were also improved significantly in gene deletion strains. Furthermore, the triplicate gene (dltD, clsA, and pssA) deletion strain was constructed, which further led to the 45.71 ± 2.43% increase of cell surface net negative charge and 26.45 ± 2.31% increase of cell membrane permeability, and the activities of nattokinase and α-amylase reached 37.15 ± 0.89 FU/mL and 305.3 ± 8.4 U/mL, increased by 46.09 ± 3.51% and 96.34 ± 7.24%, respectively. Taken together, our results confirmed that cell surface engineering via deleting dltD, clsA, and pssA is an efficient strategy for enhanced production of target proteins, and this research provided a promising host strain of B. licheniformis for efficient protein expression.

Metabolomics analysis reveals global acetoin stress response of Bacillus licheniformis.

INTRODUCTION: Acetoin serves as a high value-added platform with a broad range of applications, and can be effectively produced by Bacillus licheniformis. However, its toxicity to the producing strain hinders the higher acetoin production, and current knowledge about the acetoin resistance mechanisms of B. licheniformis is quite limited. OBJECTIVES: To comprehensively investigate the metabolic changes in B. licheniformis under acetoin stress. METHODS: We used gas chromatography-mass spectrometry based untargeted metabolomics approach to measure the metabolic profiles of B. licheniformis under 20, 40 and 80 g/L acetoin stress. Transcriptional analysis was conducted to verify the metabolomics results. RESULTS: A total of 119 metabolites were identified in our experiment. The metabolic responses of B. licheniformis to acetoin stress were as follows: (i) pentose phosphate pathway and tricarboxylic acid (TCA) cycle were negatively affected by acetoin stress. In turn, glyoxylate cycle was activated to supply malic acid. (ii) Acetoin stress induced the accumulation of serine, valine, leucine and protective osmolytes (glycine and proline). (iii) Acetoin stress induced a higher saturated fatty acid ratio, which indicated a lower fluidity of cell membrane that could inhibit the entry of acetoin into cytoplasm. (iv) Synthesis of phosphatidylserine was enhanced, and phosphatidylethanolamine content was probably increased under acetoin stress. CONCLUSIONS: This study revealed the metabolic perturbations of B. licheniformis to acetoin stress. In response to acetoin stress, glyoxylate cycle was activated, protective osmolytes were accumulated, saturated fatty acid ratio was elevated and synthesis of phosphatidylserine was enhanced in B. licheniformis.

13C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-γ-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with various applications. Teichoic acid (TA) is a special component of cell wall in gram-positive bacteria, and its D-alanylation modification can change the net negative charge of cell surface, autolysin activity and cationic binding efficiency, and might further affect metabolic production. In this research, four genes (dltA, dltB, dltC, and dltD) of dlt operon were, respectively, deleted and overexpressed in the γ-PGA producing strain Bacillus licheniformis WX-02. Our results implied that overexpression of these genes could all significantly increase γ-PGA synthetic capabilities, among these strains, the dltB overexpression strain WX-02/pHY-dltB owned the highest γ-PGA yield (2.54 g/L), which was 93.42% higher than that of the control strain WX-02/pHY300 (1.31 g/L). While, the gene deletion strains produced lower γ-PGA titers. Furthermore, 13C-Metabolic flux analysis was conducted to investigate the influence of dltB overexpression on metabolic flux redistribution during γ-PGA synthesis. The simulation data demonstrated that fluxes of pentose phosphate pathway and tricarboxylic acid cycle in WX-02/pHY-dltB were 36.41 and 19.18 mmol/g DCW/h, increased by 7.82 and 38.38% compared to WX-02/pHY300 (33.77 and 13.86 mmol/g DCW/h), respectively. The synthetic capabilities of ATP and NADPH were also increased slightly. Meanwhile, the fluxes of glycolytic and by-product synthetic pathways were all reduced in WX-02/pHY-dltB. All these above phenomenons were beneficial for γ-PGA synthesis. Collectively, this study clarified that overexpression of dltB strengthened the fluxes of PPP pathway, TCA cycle and energy metabolism for γ-PGA synthesis, and provided an effective strategy for enhanced production of γ-PGA.

Organobase catalyzed straightforward synthesis of phosphinyl functionalized 2H-pyran cores from allenylphosphine oxides and 1,3-diones.

A cost-effective system is revealed here to construct polyfunctionalized 2H-pyran cores containing phosphinyl groups with an organobase as a catalyst. Good to excellent yields were obtained under the optimized reaction conditions, with a broad substrate scope tolerated.

Enhanced production of heterologous proteins by Bacillus licheniformis with defective D-alanylation of lipoteichoic acid.

Heterologous expression is an efficient strategy for target protein production. Dlt operon plays the important role in the D-alanylation of lipoteichoic acid, which might affect the net negative charge of cell wall for protein secretion. In this study, dlt operon was deleted to improve the target protein production, and nattokinase, α-amylase and ß-mannanase with different isoelectric points (PIs) were served as the target proteins. Firstly, our results implied that deletions of dltA, dltB, dltC and dltD improved the net negative charge of cell wall for extracellular protein secretion respectively, and among which, the dltB deficient strain DW2ΔdltB showed the best performance, its nattokinase (PI: 8.60) activity was increased by 27.50% compared with that of DW2/pP43SacCNK. Then, the dltABCD mutant strain was constructed, and the net negative charge and nattokinase activity were increased by 55.57% and 37.13% respectively, due to the deficiency of dltABCD. Moreover, it was confirmed that the activities of α-amylase (PI: 6.26) and ß-mannanase (PI: 5.75) were enhanced by 44.53% and 53.06% in the dltABCD deficient strains, respectively. Collectively, this study provided a strategy that deletion of dlt operon improves the protein secretion in B. licheniformis, and which strategy was more conducive to the target protein with lower PI.

Enhanced production of poly-γ-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 µmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

Effects of different radio-opacifying agents on physicochemical and biological properties of a novel root-end filling material.

BACKGROUND/PURPOSE: Radio-opacity is an essential attribute of ideal root-end filling materials because it is important for clinicians to observe root canal filling and to facilitate the follow-up instructions. The novel root-end filling material (NRFM) has good cytocompatibility and physicochemical properties but low intrinsic radio-opacity value. To improve its radio-opacity value, three novel radio-opaque root-end filling materials (NRRFMs) were developed by adding barium sulphate (NRFM-Ba), bismuth trioxide (NRFM-Bi) and zirconium dioxide (NRFM-Zr) to NRFM, respectively. The purpose of this study was to identify the suitable radio-opacifier for NRFM through evaluating their physicochemical and biological properties, in comparison with NRFM and glass ionomer cement (GIC). METHODS: NRRFMs were characterized using X-ray diffraction (XRD) and Fourier transform infrared spectrophotometry (FTIR). Physicochemical properties including setting time, compressive strength, porosity, pH variation, solubility, washout resistance, contact angle and radiopacity were investigated. Cytocompatibility of both freshly mixed and set NRRFMs was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Alkaline phosphatase (ALP) activity assay and alizarin red staining were used to investigate the osteogenic differentiation potential of NRFM-Zr. Data were analyzed using two-way ANOVA (pH variation, solubility and ALP activity) and one-way ANOVA (for the other variables). RESULTS: (1) NRRFMs were primarily composed of hydroxyapatite, calcium carboxylate salt and the corresponding radio-opacity agents (barium sulphate, bismuth trioxide or zirconium dioxide). (2) Besides similar physicochemical properties in terms of setting time, pH variation, solubility, washout resistance and contact angle to NRFM, NRFM-Bi and NRFM-Zr exhibited lower porosity and greater compressive strength after being set for 7 days and their radio-opacity were greater than the 3 mm aluminium thickness specified in ISO 6876 (2001). (3) MTT assay revealed that freshly mixed and set NRFM-Zr presented better cell viability than NRFM-Ba and NRFM-Bi at 24 hours and 48 hours (P<0.05). (4) NRFM-Zr significantly enhanced ALP activity and calcium formation of human osteoblast-like Saos-2 cells when compared with negative group and GIC (P<0.05). CONCLUSION: NRFM-Zr presents desirable physicochemical and biological properties, thus zirconium dioxide may be a suitable radio-opacifier for NRFM.

Enhanced Production of Poly-γ-glutamic acid by Overexpression of the Global Anaerobic Regulator Fnr in Bacillus licheniformis WX-02.

Poly-γ-glutamic acid is a multi-functional biopolymer with various applications. ATP supply plays an important role in poly-γ-glutamic acid (γ-PGA) synthesis. Global anaerobic regulator Fnr plays a key role in anaerobic adaptation and nitrate respiration, which might affect ATP generation during γ-PGA synthesis. In this study, we have improved γ-PGA production by overexpression of Fnr in Bacillus licheniformis WX-02. First, the gene fnr was knocked out in WX-02, and the γ-PGA yields have no significant differences between WX-02 and the fnr-deficient strain WXΔfnr in the medium without nitrate (BFC medium). However, the γ-PGA yield of 8.95 g/L, which was produced by WXΔfnr in the medium with nitrate addition (BFCN medium), decreased by 74% compared to WX-02 (34.53 g/L). Then, the fnr complementation strain WXΔfnr/pHY-fnr restored the γ-PGA synthesis capability, and γ-PGA yield was increased by 13% in the Fnr overexpression strain WX/pHY-fnr (39.96 g/L) in BFCN medium, compared to WX/pHY300 (35.41 g/L). Furthermore, the transcriptional levels of narK, narG, and hmp were increased by 5.41-, 4.93-, and 3.93-fold in WX/pHY-fnr, respectively, which led to the increases of nitrate consumption rate and ATP supply for γ-PGA synthesis. Collectively, Fnr affects γ-PGA synthesis mainly through manipulating the expression level of nitrate metabolism, and this study provides a novel strategy to improve γ-PGA production by overexpression of Fnr.

High-level production of α-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis.

OBJECTIVES: To improve target protein production by manipulating expression levels of alanine racemase in Bacillus licheniformis. RESULTS: The gene of dal was identified to be responsible for alanine racemase function. Based on the selection marker of dal, a food-grade expression system was constructed in B. licheniformis, and effects of different dal expression levels mediated by promoters on α-amylase production were investigated. The highest α-amylase activity (155 U/ml) was obtained in BL10D/pP43SAT-PtetDal, increased by 27% compared with that of the control strain BL10/pP43SAT in tetracycline-based system (123 U/ml). Moreover, the dal transcriptional level was not correlated positively with that of amyL. CONCLUSIONS: A food-grade system for high-level production of α-amylase was constructed in B. licheniformis, revealing that expression levels of selection marker significantly affected target protein production.

Autoxidative C(sp(2))-P Formation: Direct Phosphorylation of Heteroarenes under Oxygen, Metal-Free, and Solvent-Free Conditions.

We reveal here a direct autoxidative phosphorylation of heteroarenes induced by oxygen under metal-free and solvent-free conditions. This new methodology provides an economical, operationally simple, and environmentally friendly approach toward (Het)C(sp(2))-P formation with medium to excellent yields. Heteroarenes including thiazole and quinoxaline derivatives are applicable under standard conditions, which is testified via a radical mechanism.

Cross-Coupling Hydrogen Evolution by Visible Light Photocatalysis Toward C(sp(2))-P Formation: Metal-Free C-H Functionalization of Thiazole Derivatives with Diarylphosphine Oxides.

Visible light along with 5 mol % eosin B catalyzed the first direct C-H phosphorylation of thiazole derivatives with diarylphosphine oxides by a photoredox process in the absence of an external oxidant. The scope of thiazoles and phosphine oxides was further investigated, as was functional group tolerance. The general and operational simplicity provides a novel metal and oxidant-free alternative for the formation of heteroaryl-P bonds, and only molecular hydrogen is generated as a byproduct.

The Toxicity and Detoxifying Mechanism of Cycloxaprid and Buprofezin in Controlling Sogatella furcifera (Homoptera: Delphacidae).

The effects of cycloxaprid (a modified neonicotinoid insecticide) and buprofezin (a thiadiazine insecticide) on mortality of the white-backed planthopper (WBPH), Sogatella furcifera, were determined in laboratory assays. Cycloxaprid killed WBPH nymphs and adults but buprofezin killed only nymphs, and cycloxaprid acted faster than buprofezin. One day after infestation, mortality of third-instar nymphs was >65% with cycloxaprid at 125 mg liter(-1) but was <38% with buprofezin at 148 mg liter(-1). By the 4th day after infestation, however, control of nymphs by the two insecticides was similar, and cycloxaprid at 125 mg liter(-1) caused ≥ 80% mortality of adults but buprofezin at 148 mg liter(-1) (the highest rate tested) caused almost no adult mortality. LC50 values for cycloxaprid were lowest with nymphs, intermediate with adult males, and highest with adult females. Although buprofezin was slower acting than cycloxaprid, its LC50 for nymphs 5 d after infestation was 3.79-fold lower than that of cycloxaprid. Mean carboxylesterase (CarE) specific activity of nymphal WBPH treated with cycloxaprid and buprofezin was higher than that of control, but there was no significant difference between cycloxaprid and control (no insecticide), and it was significantly higher for buprofezin than those of cycloxaprid and control. For glutathione S-transferase and mixed function oxygenase, the specific activity of nymphal WBPH treated with buprofezin was significantly higher than those of cycloxaprid and control, too.

α-Allenyl Ethers as Starting Materials for Palladium Catalyzed Suzuki-Miyaura Couplings of Allenylphosphine Oxides with Arylboronic Acids.

We disclose here the first palladium-catalyzed Suzuki-Miyaura couplings of aryl ethers functionalized allenylphosphine oxides with arylboronic acids. This new methodology with α-allenyl ethers as starting materials provides a novel approach to generate phosphinoyl 1,3-butadienes and derivatives with medium to excellent yields. The reaction tolerates a variety of functional groups to afford ranges of structurally diverse substituted phosphionyl 1,3-butadienes. For unsymmetrical allene substrates, high stereospecific additions to give E-isomers are usually observed. On the basis of the known palladium and allene chemistry, a mechanism is proposed.