Five resveratrol sulfate metabolites were synthesized and assessed for activities known to be mediated by resveratrol: inhibition of tumor necrosis factor (TNF)-α-induced NFκB activity, cylcooxygenases (COX-1 and COX-2), aromatase, nitric oxide production in endotoxin-stimulated macrophages, and proliferation of KB or MCF7 cells, induction of quinone reductase 1 (QR1), accumulation in the sub-G 1 phase of the cell cycle, and quenching of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. Two metabolites showed activity in these assays; the 3-sulfate exhibited QR1 induction, DPPH free radical scavenging, and COX-1 and COX-2 inhibitory activities, and the 4′-sulfate inhibited NFκB induction, as well as COX-1 and COX-2 activities. Resveratrol, as well as its 3′-sulfate and 4-sulfate, inhibit NO production by NO scavenging and down-regulation of iNOS expression in RAW 264.7 cells. Resveratrol sulfates displayed low antiproliferative activity and negligible uptake in MCF7 cells.

The CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of the Streptococcus pyogenes CRISPR-Cas9 system in editing the genome of Clostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal to C. cellulolyticum due to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage in C. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.

Rationale: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high-affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors, resulting in an array of biological actions on cell proliferation, migration, survival, differentiation, and motility, and therefore could mediate asthma pathogenesis.Objectives: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation. Methods: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in bronchoalveolar lavage fluids of human patients with asthma subjected to subsegmental bronchoprovocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway-specific genetically modified mice. Measurements and Main Results: Subsegmental bronchoprovocation with allergen in patients with mild asthma resulted in a remarkable increase in bronchoalveolar lavage fluid levels of LPA enriched in polyunsaturated 22:5 and 22:6 fatty acids in association with increased concentrations of ATX protein. Using a triple-allergen mouse asthma model, we showed that ATX-overexpressing transgenic mice had a more severe asthmatic phenotype, whereas blocking ATX activity and knockdown of the LPA 2 receptor in mice produced a marked attenuation of Th2 cytokines and allergic lung inflammation. Conclusions: The ATX-LPA pathway plays a critical role in the pathogenesis of asthma. These preclinical data indicate that targeting the ATX-LPA pathway could be an effective antiasthma treatment strategy.Keywords: asthma; lysophosphatidic acid; autotaxin; allergic airway inflammation supplied the ATX inhibitor, GWJ-23. V.A., E.K., and I.N. were involved in discussions related to animal dosage. A.J.M. and S.S.S. provided breeding pairs of ATX-Tg and ATX 1/2 mice. S.J.A. managed the inflammatory cell purification core lab for the SBP-AG protocol, designed experiments, interpreted data, coordinated regular scientific research meetings for the project, and edited the manuscript. V.N. conceptualized the study, designed mouse experiments, interpreted data, provided genetically modified mice, and wrote part of and edited the manuscript. J.W.C. obtained the SBP-AG IRB and IND approval, supervised mouse experiments and performance of the human SBP-AG protocol, designed experiments, interpreted and analyzed data, and edited the manuscript. All authors contributed to data discussion and review of the manuscript.Correspondence and requests for reprints should be addressed to John W. What This Study Adds to the FieldThe enzyme autotaxin (ATX) and two of its LPA products, LPA 22:5 and LPA 22:6, are markedly and selectively increased in the bronchoalveolar lavage fluid of human patients with asthma in response to airway allergen ch...

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Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(؊1) O1. R378, a residue interacting with Glc(؉1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(؊2) to Glc(؊4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(؊2) to Glc(؊4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.

Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 Å, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.innate immune ͉ tyrosinase ͉ melanization ͉ zymogen activation ͉ hemocyanin P henoloxidase (PO), a critical component of the innate immune system in insects and crustaceans, is present as a zymogen [prophenoloxidase (PPO)] in hemolymph and becomes activated on wounding or infection (1, 2). Possessing ohydroxylase (EC 1.14.18.1) and o-di-PO (EC 1.10.3.1) activities, PO converts a variety of monophenolic and o-diphenolic substrates to o-quinones (3). Quinones can act as cross-linkers for wound healing, and they also polymerize to form melanin capsules around parasites and parasitoids (4-6). Quinones and other reactive intermediates (e.g., 5,6-dihydroxyindole) directly kill microbial pathogens (7). Although PO-generated compounds are powerful weapons against pathogens, they could also cause damage to host tissues and cells. Consequently, the activation of PPO is mediated by a cascade of highly specific serine proteases and regulated as a local transient reaction against invading organisms (8).The proteolytic activation of PPO requires a trypsin-like serine protease, known as PPO activating protease (PAP) or PPO activating enzyme, which cuts the protein substrate next to an Arg residue near its amino-terminus (9-13). PAP contains regulatory clip domain(s) followed by a catalytic domain that hydrolyzes synthetic substrates at various ionic strengths and cleaves PPO in low-salt buffers preferably. In some insects, PAP generates active PO in the presence of an auxiliary factor consisting of clip-domain serine protease homolog (SPH), which lacks catalytic activity because of the substitution of the active site Ser by Gly (14,15). Unlike its precursor, active PO selfassociates into oligomers, binds to other proteins, and sticks to column matrices; consequently, it has n...

Producing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example for the development of a Clostridium cellulolyticum strain for isobutanol synthesis directly from cellulose. This strategy exploits the host's natural cellulolytic activity and the amino acid biosynthesis pathway and diverts its 2-keto acid intermediates toward alcohol synthesis. Specifically, we have demonstrated the first production of isobutanol to approximately 660 mg/liter from crystalline cellulose by using this microorganism.

BackgroundThe model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.ResultsThe first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway.ConclusionsThe efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.