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Lignin is associated with cell wall rigidity, water and solute transport, and resistance to diverse stresses in plants. Lignin consists of polymerized monolignols (p-coumaryl, coniferyl, and sinapyl alcohols), which are synthesized by cinnamyl alcohol dehydrogenase (CAD) in the phenylpropanoid pathway. We previously investigated cold-induced IbCAD1 expression by transcriptome profiling of cold-stored tuberous roots of sweetpotato (Ipomoea batatas [L.] Lam). In this study, we confirmed that IbCAD1 expression levels depended on the sweetpotato root type and were strongly induced by several abiotic stresses. We generated transgenic sweetpotato plants overexpressing IbCAD1 (TC plants) to investigate CAD1 physiological functions in sweetpotato. TC plants displayed lower root weights and lower ratios of tuberous roots to pencil roots than non-transgenic (NT) plants. The lignin contents in tuberous roots of NT and TC plants differed slightly, but these differences were not significant. By contrast, monolignol levels and syringyl (S)/guaiacyl (G) ratios were higher in TC plants than NT plants, primarily owing to syringyl unit accumulation. Tuberous roots of TC plants displayed enhanced low-temperature (4 °C) storage with lower malondialdehyde and H2O2 contents than NT plants. We propose that high monolignol levels in TC tuberous roots served as substrates for increased peroxidase activity, thereby enhancing antioxidation capacity against cold stress–induced reactive oxygen species. Increased monolignol contents and/or increased S/G ratios might contribute to pathogen-induced stress tolerance as a secondary chilling-damage response in sweetpotato. These results provide novel information about CAD1 function in cold stress tolerance and root formation mechanisms in sweetpotato.

Avermectin, produced by Streptomyces avermitilis, is an active compound protective against nematodes, insects, and mites. However, its potential usage is limited by its low aqueous solubility. The uridine diphosphate (UDP)-glycosyltransferase (BLC) from Bacillus licheniformis synthesizes avermectin glycosides with improved water solubility and in vitro antinematodal activity. However, enzymatic glycosylation of avermectin by BLC is limited due to the low conversion rate of this reaction. Thus, improving BLC enzyme activity is necessary for mass production of avermectin glycosides for field application. In this study, the catalytic activity of BLC toward avermectin was enhanced via directed evolution. Three mutants from the BLC mutant library (R57H, V227A, and D252V) had specific glucosylation activity for avermectin 2.0-, 1.8-, and 1.5-fold higher, respectively, than wild-type BLC. Generation of combined mutations via site-directed mutagenesis led to even further enhancement of activity. The triple mutant, R57H/V227A/D252V, had the highest activity, 2.8-fold higher than that of wild-type BLC. The catalytic efficiencies (Kcat/Km) of the best mutant (R57H/V227A/D252V) toward the substrates avermectin and UDP-glucose were improved by 2.71- and 2.29-fold, respectively, compared to those of wild-type BLC. Structural modeling analysis revealed that the free energy of the mutants was - 1.1 to - 7.1 kcal/mol lower than that of wild-type BLC, which was correlated with their improved activity. KEY POINTS: • Directed evolution improved the glucosylation activity of BLC toward avermectin. • Combinatorial site-directed mutagenesis led to further enhanced activity. • The mutants exhibited lower free energy values than wild-type BLC.

Convention hand polishing, which is widely used for delayering, is becoming increasingly difficult as metal lines and stacks in semiconductor devices get thinner. For one thing, endpointing at the exact targeted layer and region of interest is a major challenge. The presence of cobalt and its propensity to oxidize, thus complicating electrical measurements, is another challenge. In this study, the authors demonstrate an alternative delayering method based on plasma focused ion beam (PFIB) milling aided by DX gas. The workflow associated with the new method is more efficient than that of conventional hand polishing and can help prevent cobalt oxidation.

Virulence factors and biofilms constitute attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Among alkyl gallates, propyl gallate (PG) and octyl gallate (OG) are used as food preservatives. Here we found that alkyl gallates differentially affect virulence, biofilm formation, and quorum sensing (QS) in Pseudomonas aeruginosa. Ethyl gallate (EG), PG, and butyl gallate (BG) inhibited biofilm formation and virulence factors including elastase, pyocyanin, and rhamnolipid, in P. aeruginosa without affecting cell viability by antagonizing the QS receptors LasR and RhlR. PG exhibited the most potent activity. Interestingly, hexyl gallate (HG) inhibited the production of rhamnolipid and pyocyanin but did not affect elastase production or biofilm formation. Notably, OG inhibited the production of rhamnolipid and pyocyanin but stimulated elastase production and biofilm formation. Analysis of QS signaling molecule production and QS gene expression suggested that HG inhibited RhlR, while OG activated LasR but inhibited PqsR. This mechanism was confirmed using QS mutants. Additionally, PG prevented the virulence of P. aeruginosa in Caenorhabditis elegans and a mouse model. This is the first report of the differential effects of alkyl gallates on QS systems and PG has great potential as an inhibitor of the virulence and biofilm formation of P. aeruginosa.

Bacterial biofilms play roles in infections and avoidance of host defense mechanisms of medically important pathogens and increase the antibiotic resistance of the bacteria. Nitric oxide (NO) is reported to be involved in both biofilm formation and dispersal, which are conflicting processes. The mechanism by which NO regulates biofilm dispersal is relatively understood, but there are no reports about how NO is involved in biofilm formation. Here, by investigating the mechanism by which complestatin inhibits biofilm formation, we describe a novel mechanism for governing biofilm formation in Escherichia coli and Pseudomonas aeruginosa. Nitrite transporter is required for biofilm formation via regulation of NO levels and subsequent c-di-GMP production. Additionally, the nitrite transporter contributes more to P. aeruginosa virulence than quorum sensing. Thus, this study identifies nitrite transporters as new antibiofilm targets for future practical and therapeutic agent development., Biofilm-forming bacteria, including the Gram-negative Pseudomonas aeruginosa, cause multiple types of chronic infections and are responsible for serious health burdens in humans, animals, and plants. Nitric oxide (NO) has been shown to induce biofilm dispersal via triggering a reduction in cyclic-di-GMP levels in a variety of bacteria. However, how NO, at homeostatic levels, also facilitates biofilm formation is unknown. Here, we found that complestatin, a structural analog of vancomycin isolated from Streptomyces, inhibits P. aeruginosa biofilm formation by upregulating NO production via nitrite reductase (NIR) induction and c-di-GMP degradation via phosphodiesterase (PDE) stimulation. The complestatin protein target was identified as a nitrite transporter from a genome-wide screen using the Keio Escherichia coli knockout library and confirmed using nitrite transporter knockout and overexpression strains. We demonstrated that the nitrite transporter stimulated biofilm formation by controlled NO production via appropriate NIR suppression and subsequent diguanylate cyclase (DGC) activation, not PDE activity, and c-di-GMP production in E. coli and P. aeruginosa. Thus, this study provides a mechanism for NO-mediated biofilm formation, which was previously not understood.

Films containing microencapsulated cinnamon oil (CO) were developed using a large‐scale production system to protect against the Indian meal moth (Plodia interpunctella). CO at concentrations of 0%, 0.8%, or 1.7% (w/w ink mixture) was microencapsulated with polyvinyl alcohol. The microencapsulated CO emulsion was mixed with ink (47% or 59%, w/w) and thinner (20% or 25%, w/w) and coated on polypropylene (PP) films. The PP film was then laminated with a low‐density polyethylene (LDPE) film on the coated side. The film with microencapsulated CO at 1.7% repelled P. interpunctellamost effectively. Microencapsulation did not negatively affect insect repelling activity. The release rate of cinnamaldehyde, an active repellent, was lower when CO was microencapsulated than that in the absence of microencapsulation. Thermogravimetric analysis exhibited that microencapsulation prevented the volatilization of CO. The tensile strength, percentage elongation at break, elastic modulus, and water vapor permeability of the films indicated that microencapsulation did not affect the tensile and moisture barrier properties (P> 0.05). The results of this study suggest that effective films for the prevention of Indian meal moth invasion can be produced by the microencapsulation of CO using a large‐scale film production system. Low‐density polyethylene‐laminated polypropylene films printed with ink incorporating microencapsulated cinnamon oil using a large‐scale film production system effectively repelled Indian meal moth larvae. Without altering the tensile and moisture barrier properties of the film, microencapsulation resulted in the release of an active repellent for extended periods with a high thermal stability of cinnamon oil, enabling commercial film production at high temperatures. This anti‐insect film system may have applications to other food‐packaging films that use the same ink‐printing platform.

Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP)-glycosyltransferase from Bacillus licheniformis with various UDP sugars. As a result, the following four avermectin B1a glycosides were produced: avermectin B1a 4″-β-D-glucoside, avermectin B1a 4″-β-D-galactoside, avermectin B1a 4″-β-L-fucoside, and avermectin B1a 4″-β-2-deoxy-D-glucoside. The avermectin B1a glycosides were structurally analyzed based on HR-ESI MS and 1D and 2D nuclear magnetic resonance spectra, and the anti-nematodal effect of avermectin B1a 4″-β-D-glucoside was found to exhibit the highest activity (IC50 = 0.23 μM), which was approximately 32 times greater than that of avermectin B1a (IC50 = 7.30 μM), followed by avermectin B1a 4″-β-2-deoxy-D-glucoside (IC50 = 0.69 μM), avermectin B1a 4″-β-L-fucoside (IC50 = 0.89 μM), and avermectin B1a 4″-β-D-galactoside (IC50 = 1.07 μM). These results show that glycosylation of avermectin B1a effectively enhances its in vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.

FtsZ, a bacterial cell-division protein, is an attractive antibacterial target. In the screening for an inhibitor of Staphylococcus aureus FtsZ, madurahydroxylactone (1) and its related derivatives 2-5 were isolated from Nonomuraea sp. AN100570. Compound 1 inhibited S. aureus FtsZ with an IC50 of 53.4 μM and showed potent antibacterial activity against S. aureus and MRSA with an MIC of 1 μg/ml, whereas 2-5 were weak or inactive. Importantly, 1 induced cell elongation in the cell division phenotype assay, whereas 2-5 did not. It indicates that 1 exhibits its potent antibacterial activity via inhibition of FtsZ, and the hydroxyl group and hydroxylactone ring of 1 are critical for the activity. Thus, madurahydroxylactone is a new type of inhibitor of FtsZ.

Screening for new natural anti-neuroinflammatory compounds was performed with the traditional folk medicine Genkwa Flos, which potently inhibited nitric oxide (NO) production by LPS-activated microglial BV-2 cells. Two new lathyrane-type diterpenes, genkwalathins A (1) and B (2), and 14 known daphnane-type diterpenes (3–16) were isolated. The lathyrane-type diterpenes were isolated for the first time from the Thymelaeaceae family in this study. Compounds 1 and 2 moderately inhibited LPS-induced NO production in BV-2 cells without affecting cell viability, while six daphnane-type diterpenes (3, 4, 6, 7, 9 and 10) potently reduced NO production with IC50 values less than 1 μM, although they did display weak cytotoxicity. A structure–activity relationship study on the daphnane-type diterpenes indicated that the stereochemistry at C-19, the benzoate group at C-20, and the epoxide moiety could be important for their anti-neuroinflammatory effects.

Menthol has a considerable cooling effect, but the use range of menthol is limited because of its extremely low solubility in water and inherent flavor. (−)-Menthol β-glucoside was determined to be more soluble in water (>27 times) than (−)-menthol α-glucoside; hence, β-anomer-selective glucosylation of menthol is necessary. The in vitro glycosylation of (−)-menthol by uridine diphosphate glycosyltransferase (BLC) from Bacillus licheniformis generated (−)-menthol β-glucoside and new (−)-menthol β-galactoside and (−)-menthol N-acetylglucosamine. The maximum conversion rate of menthol to (−)-menthol β-d-glucoside by BLC was found to be 58.9%. Importantly, (−)-menthol β-d-glucoside had a higher cooling effect and no flavor compared with menthol. In addition, (−)-menthol β-d-glucoside was determined to be a non-sensitizer in a skin allergy test in the human cell line activation test, whereas menthol was a sensitizer. Electronic supplementary material The online version of this article (doi:10.1186/s13568-017-0468-0) contains supplementary material, which is available to authorized users.

The methanol extract of the roots and stems of Daphne genkwa and its constituents yuanhuacin (1) and genkwanine N were previously reported to have Nurr1 activating effects and neuroprotective effects in an animal model of Parkinson's disease (PD). In this study, four more daphnane-type diterpenes (acutilonine F (2), wikstroemia factor M1 (3), yuanhuadine (5), and yuanhuatine (6)) and two phorbol-type diterpenes (prostratin Q (4) and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)) were isolated as Nurr1 activating compounds from the D. genkwa extract. Consistent with their higher Nurr1 activating activity, compounds 1, 4, 5, and 7 exhibited higher inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial BV-2 cells with an IC50 (µM) of 1-2, which was 15-30 times more potent than that of minocycline (29.9 µM), a well-known anti-neuroinflammatory agent. Additionally, these diterpenes reduced expression and transcription of LPS-induced pro-inflammatory cytokines in BV-2 cells. Thus, the daphnane-type and phorbol-type diterpenes had anti-neuroinflammatory activity with Nurr1 activation and could be responsible for the anti-PD effect of the roots and stems of D. genkwa.

A new microbial decontamination system combining washing with a natural antimicrobial solution and coating with a carnauba wax (CW)-based antimicrobial coating was developed and its effects on mandarin storability were investigated. Mandarins were washed with an antimicrobial solution and/or coated with grapefruit seed extract-CW (GSE/CW). Values for the disease incidence of Penicillium digitatum in untreated mandarins; mandarins coated with GSE/CW without washing; and mandarins coated with GSE/CW after washing with a fumaric acid (FA) solution of slightly acidic electrolyzed water, a highly activated calcium oxide (CaO) aqueous solution, or CaO solution followed by FA solution were 96.0, 70.0, 78.8, 50.0, and 72.2%, respectively. GSE/CW coating after CaO washing was most effective in inhibiting P. digitatum growth during storage at 25 °C. Compared to untreated samples, GSE/CW coating alone or after CaO washing retained CO2 generation, firmness, and total polyphenol content of mandarins at 25 °C. Such treatments also effectively maintained mandarin pH, ascorbic acid concentration, and antioxidant capacity at both 4 and 25 °C. Moreover, GSE/CW coating after CaO washing more effectively inhibited P. digitatum growth at 25 °C and maintained ascorbic acid concentration and antioxidant capacity at 4 and 25 °C than GSE/CW coating alone. The microbial decontamination system integrating CaO washing and GSE/CW coating demonstrates potential for improving mandarin storability by inhibiting P. digitatum growth and improving the preservation of quality properties and sensory characteristics. PRACTICAL APPLICATION: This is the first study to develop a microbial decontamination system involving both washing with a natural antimicrobial solution and carnauba wax coating containing grapefruit seed extract to improve the storability of fruits. This system demonstrated a primary effect of inhibiting fungi that cause mandarin surface decay at 25 °C via the highly activated calcium oxide wash and secondary effects of delaying quality degradation and inhibiting fungal growth by the action of the antimicrobial coating. These effects led to improvements in mandarin storability, along with enhanced visual appeal while not affecting taste, flavor, or texture.

Carbon black particles surrounded by copper nanoparticles (Cu NPs) were synthesized using electroless plating method. Palladium chloride was adsorbed onto carbon black, followed by the reduction of palladium chloride for catalyzing the reduction of Cu ions on the surface of carbon black particles. After that, carbon black particles doped by palladium catalyst were dispersed and stirred in Cu plating bath. Cu ions being reduced, Cu NPs surrounded the surface of carbon black particles (Cu@CB). The ratios of Cu to carbon black were controlled through variation of weight of Cu ions in Cu plating bath from 1:1 to 1:7. Cu@CB was applied to electrically conductive substrates with ethyl cellulose binder. Electrical properties and morphology were measured and compared with different weight ratio of Cu and carbon black. It was found that when weight ratio of Cu to carbon black was above three, resistivity of conductive substrates fabricated decreased dramatically. Lowest resistivity was 5.93 × 10−4 Ωcm, confirming the advantages of Cu@CB which has possibility of lowering weight percentage of metal in conductive substrates through simple process.

This study investigated the effects of washing and packaging combined treatments on satsuma mandarin (Citrus unshiu Marc.) quality during storage at 4 °C and 25 °C. The treatments involved the combinations of washing using calcium oxide (CaO, 0.2%) solution, carnauba wax coating with grapefruit seed extract (1%) (CW coating), and modified atmosphere packaging (MAP) using O2–CO2–N2 (9.9%, 2.1%, and 88.0%, respectively). The growth of P. digitatum was inhibited across all samples at 4 °C and 25 °C. CaO washing delayed the increase in mandarin CO2 generation during storage at both temperatures and the decrease in antioxidant capacity at 4 °C. CW coating retarded the increase in CO2 generation and the decrease in firmness and antioxidant capacity at both temperatures. MAP delayed the decrease in ascorbic acid concentration at 25 °C and the increase in CO2 generation at 4 °C and 25 °C and also retarded the reduction in antioxidant capacity with or without CaO washing at both temperatures. The combination of three treatments effectively delayed decreases in firmness, ascorbic acid concentration, and antioxidant capacity, and retarded increases in the CO2 generation of mandarins during storage, which demonstrates the potential effectiveness of combined treatments in extending the shelf life of mandarins.

Seven new coralmycin derivatives, coralmycins C (1), D (2), E (3), F (4), G (5), H (6), and I (7), along with three known compounds, cystobactamids 891-2 (8), 905-2 (9), and 507 (10), were isolated from a large-scale culture of the myxobacteria Corallococcus coralloides M23. The structures of these compounds, including their relative stereochemistries, were elucidated by interpretation of their spectroscopic and CD data. The structure-activity relationships of their antibacterial and DNA gyrase inhibitory activities indicated that the para-nitrobenzoic acid unit is critical for the inhibition of DNA gyrase and bacterial growth, while the nitro moiety of the para-nitrobenzoic acid unit and the isopropyl chain at C-4 could be important for permeability into certain Gram-negative bacteria, including Pseudomonas aeruginosa and Klebsiella pneumoniae, and the &beta, methoxyasparagine moiety could affect cellular uptake into all tested bacteria. These results could facilitate the chemical optimization of coralmycins for the treatment of multidrug-resistant Gram-negative bacteria.

The heterologous expression of the biosynthetic gene cluster (BGC) of natural products enables the production of complex metabolites in a well-characterized host, and facilitates the generation of novel analogues by the manipulation of the genes. However, the BGCs of glycopeptides such as vancomycin, teicoplanin, and complestatin are usually too large to be directly cloned into a single cosmid. Here, we describe the heterologous expression of the complestatin BGC. The 54.5 kb cluster was fully reconstituted from two overlapping cosmids into one cosmid by λ-RED recombination-mediated assembly. Heterologous expression of the assembled gene cluster in Streptomyces lividans TK24 resulted in the production of complestatin. Deletion of cytochrome P450 monooxygenase genes (open reading frames 10 and 11) and heterologous expression of the modified clusters led to the production of two new monocyclic and linear derivatives, complestatins M55 and S56.

Surface-enhanced Raman scattering (SERS) imaging has been used for the targeting and imaging of specific cancer markers in live cells. For this purpose, Au/Ag core-shell nanoparticles, conjugated with monoclonal antibodies, were prepared. The procedures to label live cells with those bimetallic nanoprobes have been developed and used for highly sensitive SERS imaging of live cells. In the present study, live HEK293 cells expressing PLCgamma1 have been used as the optical imaging target. Our results demonstrate the potential feasibility of SERS imaging technology for the highly sensitive imaging of cancer biomarkers in live cells.

In this study, receptor binding profiles of opioid ligands for subtypes of opioid δ-receptors were examined employing [ 3 H]D-Pen 2 ,D-Pen 5 -enkephalin ([ 3 H]DPDPE) and [ 3 H]Ile 5,6 -deltorphin II ([ 3 H]Ile-Delt II) in human cerebral cortex membranes. [ 3 H]DPDPE, a representative ligand for δ 1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 ± 0.21 nM and maximal binding capacity (Bmax) value of 20.78 ± 3.13 fmol/mg protein. Homologous competition curve of [ 3 H]Ile-Delt II, a representative ligand for δ 2 sites, was best fit by the one-site model (Kd = 0.82 ± 0.07 nM). Bmax value (43.65 ± 2.41 fmol/mg) for [ 3 H]Ile-Delt II was significantly greater than that for [ 3 H]DPDPE. DPDPE, [D-Ala 2 ,D-Leu 5 ]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [ 3 H]DPDPE than for those of [ 3 H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser 2 ,Leu 5 ,Thr 6 ]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [ 3 H]DPDPE and [ 3 H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid δ-receptors, δ 1 and δ 2 , exist in human cerebral cortex with different ligand binding profiles.

A novel fluorescent sensor based on the Zn2+-DPA complex shows selective detection of flavin adenine dinucleotide (FAD) from among other flavins in water, and it was used to detect FAD in eosinophils by fluorescence microscopy and FACS.

We report here the results on N-acetyl-L-proline-N'-methylamide (Ac-Pro-NHMe) calculated at the HF/6-31+G(d) level with the conductor-like polarizable continuum model (CPCM) of self-consistent reaction field methods to investigate the changes of backbone and prolyl ring along the cis-trans isomerization of the prolyl peptide bond. From the potential energy surface, the barrier to ring flip from the down-puckered conformation to the up-puckered one is estimated to be 2.5 and 3.2 kcal/mol for trans and cis conformers of Ac-Pro-NHMe, respectively. In particular, the ring flip seems to be inaccessible in the intermediate regions between trans and cis conformations, because of higher barriers (approximately 13-19 kcal/mol) to rotation of the prolyl peptide bond. The torsion angles for backbone and prolyl ring vary largely around the transition states at omega' approximately 120 degrees and -70 degrees for the prolyl peptide bond. Three kinds of puckering amplitudes show the same trend of puckering along the cis-trans isomerization although their absolute values are different. In particular, trans and cis conformations have the almost same degree of puckering. The cis populations and barriers to rotation of the prolyl peptide bond for Ac-Pro-NHMe are increased with the increase of solvent polarity, which is mainly ascribed to the decreases of relative free energies for cis conformations and the increase of relative free energies for transition states.

Background and Purpose —Angiopoietin-1 (Ang1) is a vasculogenic factor that signals through the endothelial cell–specific Tie2 receptor tyrosine kinase. We recently reported that Ang1 prevented apoptosis induced by serum deprivation in endothelial cells. In this study, we examined whether Ang1 prevents apoptosis in endothelial cells treated with irradiation or clinical concentrations of mannitol. Methods and Results —Ang1 prevented irradiation- and mannitol-induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the antiapoptotic effect of Ang1. Two phosphatidylinositol 3′-kinase (PI3-kinase)–specific inhibitors, wortmannin and LY294002, blocked the Ang1-induced antiapoptotic effect. The antiapoptotic potency of Ang1 was similar to or greater than that of vascular endothelial growth factor, basic fibroblast growth factor, and endothelin-1. Ang1 also prevented apoptosis in cultured endothelial cells from porcine pulmonary and coronary arteries and in endothelial cells of explanted rat aorta. Conclusions —Ang1 promotes the survival of endothelial cells in irradiation- and mannitol-induced apoptosis through Tie2 receptor binding and PI3-kinase activation. Pretreatment with Ang1 could be beneficial in maintaining normal endothelial cell integrity during intracoronary irradiation or systemic mannitol therapy.