Specialized metabolism by trichome-enriched Rubisco and fatty acid synthase components.

Acylsugars, specialized metabolites with defense activities, are secreted by trichomes of many solanaceous plants. Several acylsugar metabolic genes (AMGs) remain unknown. We previously reported multiple candidate AMGs. Here, using multiple approaches, we characterized additional AMGs. First, we identified differentially expressed genes between high- and low-acylsugar-producing F2 plants derived from a cross between cultivated tomato (Solanum lycopersicum) and a wild relative (Solanum pennellii), which produce acylsugars that are ∼1% and ∼20% of leaf dry weight, respectively. Expression levels of many known and candidate AMGs positively correlated with acylsugar amounts in F2 individuals. Next, we identified lycopersicum-pennellii putative orthologs with higher nonsynonymous to synonymous substitutions. These analyses identified four candidate genes, three of which showed enriched expression in stem trichomes compared to underlying tissues (shaved stems). Virus-induced gene silencing confirmed two candidates, Sopen05g009610 [beta-ketoacyl-(acyl-carrier-protein) reductase; fatty acid synthase component] and Sopen07g006810 (Rubisco small subunit), as AMGs. Phylogenetic analysis indicated that Sopen05g009610 is distinct from specialized metabolic cytosolic reductases but closely related to two capsaicinoid biosynthetic reductases, suggesting evolutionary relationship between acylsugar and capsaicinoid biosynthesis. Analysis of publicly available datasets revealed enriched expression of Sopen05g009610 orthologs in trichomes of several acylsugar-producing species. Similarly, orthologs of Sopen07g006810 were identified as solanaceous trichome-enriched members, which form a phylogenetic clade distinct from those of mesophyll-expressed "regular" Rubisco small subunits. Furthermore, δ13C analyses indicated recycling of metabolic CO2 into acylsugars by Sopen07g006810 and showed how trichomes support high levels of specialized metabolite production. These findings have implications for genetic manipulation of trichome-specialized metabolism in solanaceous crops.

Polypeptide organic radical batteries.

In only a few decades, lithium-ion batteries have revolutionized technologies, enabling the proliferation of portable devices and electric vehicles1, with substantial benefits for society. However, the rapid growth in technology has highlighted the ethical and environmental challenges of mining lithium, cobalt and other mineral ore resources, and the issues associated with the safe usage and non-hazardous disposal of batteries2. Only a small fraction of lithium-ion batteries are recycled, further exacerbating global material supply of strategic elements3-5. A potential alternative is to use organic-based redox-active materials6-8 to develop rechargeable batteries that originate from ethically sourced, sustainable materials and enable on-demand deconstruction and reconstruction. Making such batteries is challenging because the active materials must be stable during operation but degradable at end of life. Further, the degradation products should be either environmentally benign or recyclable for reconstruction into a new battery. Here we demonstrate a metal-free, polypeptide-based battery, in which viologens and nitroxide radicals are incorporated as redox-active groups along polypeptide backbones to function as anode and cathode materials, respectively. These redox-active polypeptides perform as active materials that are stable during battery operation and subsequently degrade on demand in acidic conditions to generate amino acids, other building blocks and degradation products. Such a polypeptide-based battery is a first step to addressing the need for alternative chemistries for green and sustainable batteries in a future circular economy.

Probing the Role of N-Acetyl-glutamyl 5-Phosphate, an Acyl Phosphate, in the Construction of the Azabicycle Moiety of the Azinomycins.

The azinomycins are potent antitumor agents produced by the soil bacterium Streptomyces sahachiroi and contain a novel aziridino[1,2-a]pyrrolidine core; its synthesis involves at least 14 steps. This study reports the first reconstitution of N-acetylglutamine semialdehyde formation by two enzymes encoded in the azinomycin biosynthetic gene cluster. The reaction proceeds through the formation of an acylphosphate and establishes N-acetyl-glutamyl 5-phosphate and N-acetylglutamine semialdehyde as intermediates in the complex biosynthesis of the aziridino[1,2-a]pyrrolidine moiety.

Preparation and in vitro antimicrobial activity of silver-bearing degradable polymeric nanoparticles of polyphosphoester-block-poly(L-lactide).

The development of well-defined polymeric nanoparticles (NPs) as delivery carriers for antimicrobials targeting human infectious diseases requires rational design of the polymer template, an efficient synthetic approach, and fundamental understanding of the developed NPs, e.g., drug loading/release, particle stability, and other characteristics. Herein, we developed and evaluated the in vitro antimicrobial activity of silver-bearing, fully biodegradable and functional polymeric NPs. A series of degradable polymeric nanoparticles (dNPs), composed of phosphoester and L-lactide and designed specifically for silver loading into the hydrophilic shell and/or the hydrophobic core, were prepared as potential delivery carriers for three different types of silver-based antimicrobials-silver acetate or one of two silver carbene complexes (SCCs). Silver-loading capacities of the dNPs were not influenced by the hydrophilic block chain length, loading site (i.e., core or shell), or type of silver compound, but optimization of the silver feed ratio was crucial to maximize the silver loading capacity of dNPs, up to ca. 12% (w/w). The release kinetics of silver-bearing dNPs revealed 50% release at ca. 2.5-5.5 h depending on the type of silver compound. In addition, we undertook a comprehensive evaluation of the rates of hydrolytic or enzymatic degradability and performed structural characterization of the degradation products. Interestingly, packaging of the SCCs in the dNP-based delivery system improved minimum inhibitory concentrations up to 70%, compared with the SCCs alone, as measured in vitro against 10 contemporary epidemic strains of Staphylococcus aureus and eight uropathogenic strains of Escherichia coli. We conclude that these dNP-based delivery systems may be beneficial for direct epithelial treatment and/or prevention of ubiquitous bacterial infections, including those of the skin and urinary tract.

Cultivation of lipid-producing bacteria with lignocellulosic biomass: effects of inhibitory compounds of lignocellulosic hydrolysates.

Lignocellulosic biomass has been recognized as a promising feedstock for the fermentative production of biofuel. However, the pretreatment of lignocellulose generates a number of by-products, such as furfural, 5-hydroxylmethyl furfural (5-HMF), vanillin, vanillic acids and trans-p-coumaric acid (TPCA), which are known to inhibit microbial growth. This research explores the ability of Rhodococcus opacus PD630 to use lignocellulosic biomass for production of triacylglycerols (TAGs), a common lipid raw material for biodiesel production. This study reports that R. opacus PD630 can grow well in R2A broth in the presence of these model inhibitory compounds while accumulating TAGs. Furthermore, strain PD630 can use TPCA, vanillic acid, and vanillin as carbon sources, but can only use TPCA and vanillic acid for TAG accumulation. Strain PD630 can also grow rapidly on the hydrolysates of corn stover, sorghum, and grass to accumulate TAGs, suggesting that strain PD630 is well-suited for bacterial lipid production from lignocellulosic biomass.

The genetic incorporation of thirteen novel non-canonical amino acids.

Thirteen novel non-canonical amino acids were synthesized and tested for suppression of an amber codon using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair. Suppression was observed with varied efficiencies. One non-canonical amino acid in particular contains an azide that can be applied for site-selective protein labeling.

Development of a Vinyl Ether-Functionalized Polyphosphoester as a Template for Multiple Postpolymerization Conjugation Chemistries and Study of Core Degradable Polymeric Nanoparticles.

A novel polyphosphoester (PPE) with vinyl ether side chain functionality was developed as a versatile template for postpolymerization modifications, and its degradability and biocompatibility were evaluated. An organo-catalyzed ring-opening polymerization of ethylene glycol vinyl ether-pendant cyclic phosphotriester monomer allowed for construction of poly(ethylene glycol vinyl ether phosphotriester) (PEVEP). This vinyl ether-functionalized PPE scaffold was coupled with hydroxyl- or thiol-containing model small molecules via three different types of conjugation chemistries-thiol-ene "click" reaction, acetalization, or thio-acetalization reaction-to afford modified polymers that accommodated either stable thio-ether or hydrolytically labile acetal or thio-acetal linkages. Amphiphilic diblock copolymers of poly(ethylene glycol) and PEVEP formed well-defined micelles with a narrow and monomodal size distribution in water, as confirmed by dynamic light scattering (DLS), transmission electron microscopy, and atomic force microscopy. The stability of the micelles and the hydrolytic degradability of the backbone and side chains of the PEVEP block segment were assessed by DLS and nuclear magnetic resonance spectroscopy (1H and 31P), respectively, in aqueous buffer solutions at pH values of 5.0 and 7.4 and at temperatures of 25 and 37 °C. The hydrolytic degradation products of the PEVEP segments of the block copolymers were then identified by electrospray ionization, gas chromatography, and matrix-assisted laser desorption/ionization mass spectrometry. The parent micelles and their degradation products were found to be non-cytotoxic at concentrations up to 3 mg/mL, when evaluated with RAW 264.7 mouse macrophages and OVCAR-3 human ovarian adenocarcinoma cells.

The molecular basis of sugar sensing in Drosophila larvae.

Evaluation of food chemicals is essential to make appropriate feeding decisions. The molecular genetic analysis of Gustatory receptor (Gr) genes and the characterization of the neural circuits that they engage has led to a broad understanding of taste perception in adult Drosophila [1, 2]. For example, eight relatively highly conserved members of the Gr gene family (Gr5a, Gr61a, and Gr64a-f), referred to as sugar Gr genes, are thought to be involved in sugar taste in adult flies [3-8], while the majority of the remaining Gr genes are likely to encode bitter taste receptors [9-11], albeit some function as pheromone [12-14] and carbon dioxide [15, 16] receptors. In contrast to the adult fly, relatively little is known about the cellular and molecular basis of taste perception in larvae. Here, we identify Gr43a, which was recently shown to function as a hemolymph fructose sensor in adult flies [17], as the major larval sugar receptor. We show that it is expressed in taste neurons, proventricular neurons, as well as sensory neurons of the brain. Larvae lacking Gr43a fail to sense sugars, while larvae mutant for all eight sugar Gr genes exhibit no obvious defect. Finally, we show that brain neurons are necessary and sufficient for sensing all main dietary sugars, which probably involves a postingestive mechanism of converting carbohydrates into fructose.

Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA(Pyl) pairs in the Escherichia coli BL21(DE3) cell strain.

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA(Pyl) pairs and cross recognition between nonsense codons and various tRNA(Pyl) anticodons in the Escherichia coli BL21(DE3) cell strain are reported. tRNA(CUA)(Pyl) is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N(ε)-tert-butoxycarbonyl-L-lysine (BocK). Similar to tRNA(CUA)(Pyl), tRNA(UUA)(Pyl) is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA)(Pyl) is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA)(Pyl) pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS-tRNA(UCA)(Pyl) pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU)(Pyl) fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.

Biodegradation of triclosan by a wastewater microorganism.

Triclosan, a synthetic antimicrobial agent, has been considered as an emerging environmental contaminant. Here we reported a triclosan-degrading wastewater bacterial isolate, Sphingopyxis strain KCY1, capable of dechlorinating triclosan with a stoichiometric release of chloride. The stain can degrade diphenyl ether but not 2,4,4'-tribromodiphenyl ether and 2,2',4,4'-tetrabromodiphenyl ether, despite all these three compounds are structurally similar to triclosan. While strain KCY1 was unable to grow on triclosan and catechol, it could grow with glucose, sodium succinate, sodium acetate, and phenol. When grown with complex nutrient medium containing a trace amount of triclosan (as low as 5 µg/L), the strain could retain its degradation ability toward triclosan. The maximum-specific triclosan degradation rate (q(m)) and the half-velocity constant (K(m)) are 0.13 mg-triclosan/mg-protein/day and 2.8 mg-triclosan/L, respectively. As triclosan degradation progressed, five metabolites were identified and these metabolites continue to transform into non-chlorinated end products, which was supported by a sharp drop in androgenic potential. The activity of catechol 2,3-dioxygenase in the cell extract was detected. No triclosan degradation was observed in the presence of 3-fluorocatechol, an inhibitor of meta-cleavage enzyme, suggesting that triclosan degradation proceed via meta-cleavage pathway. Based on all the observations, a degradation pathway for triclosan by strain KCY1 was proposed.

Mineralization of acephate, a recalcitrant organophosphate insecticide is initiated by a pseudomonad in environmental samples.

An aerobic bacterium capable of breaking down the pesticide acephate (O,S-dimethyl acetyl phosphoramidothioic acid) was isolated from activated sludge collected from a pesticide manufacturing facility. A phylogenetic tree based on the 16 S rRNA gene sequence determined that the isolate lies within the Pseudomonads. The isolate was able to grow in the presence of acephate at concentrations up to 80 mM, with maximum growth at 40 mM. HPLC and LC-MS/MS analysis of spent medium from growth experiments and a resting cell assay detected the accumulation of methamidophos and acetate, suggesting initial hydrolysis of the amide linkage found between these two moieties. As expected, the rapid decline in acephate was coincident with the accumulation of methamidophos. Methamidophos concentrations were maintained over a period of days, without evidence of further metabolism or cell growth by the cultures. Considering this limitation, strains such as described in this work can promote the first step of acephate mineralization in soil microbial communities.

Infrared laser-assisted desorption electrospray ionization mass spectrometry.

We have used an infrared laser for desorption of material and ionization by interaction with electrosprayed solvent. Infrared laser-assisted desorption electrospray ionization (IR LADESI) mass spectrometry was used for the direct analysis of water-containing samples under ambient conditions. An ion trap mass spectrometer was modified to include a pulsed Er:YAG laser at 2.94 microm wavelength coupled into a germanium oxide optical fiber for desorption at atmospheric pressure and a nanoelectrospray source for ionization. Analytes in aqueous solution were placed on a stainless steel target and irradiated with the pulsed IR laser. Material desorbed and ablated from the target was ionized by a continuous stream of charged droplets from the electrosprayed solvent. Peptide and protein samples analyzed using this method yield mass spectra similar to those obtained by conventional electrospray. Blood and urine were analyzed without sample pretreatment to demonstrate the capability of IR LADESI for direct analysis of biological fluids. Pharmaceutical products were also directly analyzed. Finally, the role of water as a matrix in the IR LADESI process is discussed.

Desorption electrospray ionization of aerosol particles.

We have applied desorption electrospray ionization to aerosol particles. Ions were formed from aerosols by merging suspended dry particles with an electrospray of solvent in a modified ion trap mass spectrometer. Dry aerosol particles were generated using a fluidized bed powder disperser and directed toward the inlet of the mass spectrometer. A nanospray source was used to create a spray of solvent droplets directed at the inlet and at a right angle with respect to the aerosol. Ions generated by the interaction of the particles and electrospray were transferred into the ion trap mass spectrometer. Using this method, pure samples of caffeine and erythromycin A were analyzed. In addition, commonly available food and drug powders including instant cocoa powder, artificial sweetener and ibuprofen were analyzed.

Separation and detection of individual submicron particles by capillary electrophoresis with laser-light-scattering detection.

Separation and detection of individual submicron polystyrene spheres using capillary electrophoresis with laser-light-scattering detection has been demonstrated. Electrophoretically separated particles were passed through a focused laser beam and light scattered from individual particles was collected at 90 degrees. Each diameter of polystyrene spheres injected (from 110 to 992 nm) resulted in the observation of a well-defined migration window containing multiple peaks, each arising from the light scattered by an individual particle. The migration time window for individual particles of a particular size corresponded well to the migration time of a peak from a population of particles of the same size detected using a UV absorbance detector. The electrophoretic mobility and scattered light intensity were determined for each particle detected. The average scattered light intensity for each particle size was consistent with Mie scattering theory. Particles as small as 110 nm in diameter were detected individually using this method, but particles with a diameter of 57 nm could not be individually detected. The number of single particle scattering events was counted and compared to the theoretical number of particles injected electrokinetically, and the detection efficiency determined ranged from 38 to 57% for polystyrene spheres of different sizes. The laser-light-scattering detection method was directly compared to laser-induced fluorescence detection using fluorescent polystyrene microspheres. The number of particles detected individually by each method was in agreement.

Laser ablation construction of on-column reagent addition devices for capillary electrophoresis.

A simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis. This technique uses laser ablation with the second harmonic of a Nd:YAG laser (532 nm) at 13.5 mJ/pulse and a repetition rate of 15 Hz to create these gaps. A capillary is glued to a microscope slide and positioned at the focal point of a cylindrical lens using the focused beam from a laser pointer as a reference. Gaps of 14.0 +/- 2.2 microm (n = 33) at the bore of the capillary are produced with a success rate of 94% by ablation with 400 pulses. This simple method of gap construction requires no micromanipulation under a microscope, hydrofluoric acid etching, or use of column fittings. These structures have been used for reagent addition for postcolumn derivatization with laser-induced fluorescence detection and have been tested for the separation of proteins and amino acids. Detection limits of 6 x 10(-7) and 1 x 10(-8) M have been obtained for glycine and tranferrin, respectively. Separation efficiencies obtained using these gap reactors range from 38,000 to 213,000 theoretical plates.