Genomics of Aerobic Cellulose Utilization Systems in Actinobacteria

Cellulose degrading enzymes have important functions in the biotechnology industry, including the production of biofuels from lignocellulosic biomass. Anaerobes including Clostridium species organize cellulases and other glycosyl hydrolases into large complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems comprised of independently acting enzymes, often with carbohydrate binding domains. Numerous actinobacterial genomes have become available through the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. We identified putative cellulose-degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms were tested in several assays for cellulose degradation, and eight of the organisms showed evidence of cellulase activity. The three with the highest cellulase activity were Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica. Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter are regulated by the transcriptional regulator CelR. Nine organisms appear to use the CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR also regulates cellulases, while cellulases are controlled by different regulatory sites in three organisms. Mining of genome data for cellulose degradative enzymes followed by experimental verification successfully identified several actinobacteria species which were not previously known to degrade cellulose as cellulolytic organisms

The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation

Reduction in Local Ozone Levels in Urban São Paulo Due to a Shift from Ethanol to Gasoline Use

It has been proposed that lower NOx emission fuels such as ethanol can mitigate air pollution from vehicles burning oil-based hydrocarbons. Yet, existing modeling and laboratory studies, even those seeking to simulate the same environment, vary in their predictions of how gasoline/ethanol blends affect atmospheric pollutant concentrations, including ozone. Importantly, ambient concentrations have not been evaluated during an actual – as opposed to hypothetical – shift in fuel mix in a real-world environment. Here, we report the first such study, for the subtropical megacity of São Paulo, Brazil. We combine detailed street-hour level data on regulated pollutant concentrations, meteorology, and traffic with fuel shares from a consumer demand model to compare concentrations across subsamples that differ only in the fuel mix but are otherwise similar in meteorology, anthropogenic activity, and biogenic emissions. As the gasoline share of the bi-fuel light-duty vehicle fleet rose by 62 percentage points, we estimate a robust and statistically significant reduction of about 20% in ozone concentrations, and less precise increases in NO and CO concentrations. We propose that our “model-free” analysis potentially accounts for the interaction between anthropogenic and biogenic emissions and caution that successful strategies against ozone pollution require knowledge of the local chemistry and analysis beyond the presently monitored pollutants, most notably fine particles

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.Biological and Environmental Research/[DE-FC02-07ER64494]/BER/Estados UnidosNational Science Foundation/[DGE-1256259]/NSF/Estados UnidosNational Science Foundation/[DEB-0747002]/NSF/Estados UnidosNational Science Foundation/[MCB-0702025]/NSF/Estados UnidosNational Institutes of Health/[T32 GM07215]/NIH/Estados UnidosUniversidad de Costa Rica/[]/UCR/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[]/MICITT/Costa RicaUniversity of Wisconsin-Madison's Hilldale Undergraduate Faculty Research Fellowship/[]//Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Simple and Accurate Determination of Reactivity Ratios Using a Nonterminal Model of Chain Copolymerization

We propose a new method for the determination of reactivity ratios based on a nonterminal model of copolymerization kinetics. Within the context of this model, we derive simple, reactivity-ratio-dependent expressions whose solution relies solely on monomer consumption information spanning the full range of conversion. Utilizing this method, reactivity ratios are obtained for the aluminum chelate-catalyzed copolymerization of phenyl glycidyl ether and allyl glycidyl ether (rPGE = 1.56 ± 0.01 and rAGE = 0.66 ± 0.03) with monomer consumption monitored by in situ 1H NMR spectroscopy. Additionally, this approach is applied to experimental data extracted from the literature for other copolymerization systems encompassing a range of monomer types (styrenics, isoprene, lactones, lactide, and other cyclic ethers) and polymerization type (anionic, coordination, and zwitterionic) to obtain reactivity ratios under the mechanistic assumption of nonterminal model copolymerization kinetics. We present the nonterminal model of copolymerization as the first method that should be utilized before more complex frameworks (e.g., terminal or penultimate model of chain copolymerization) are used to understand copolymerization kinetics

Monitoring multicomponent transport using in situ ATR FTIR spectroscopy

Membranes are a critical component of many energy generation and storage technologies, including artificial photosynthesis systems that reduce atmospheric CO2 to high-value products. In this study, we used in situ ATR FTIR spectroscopy to monitor the crossover of three commonly-reported CO2 reduction products—methanol, sodium formate, and sodium acetate—through Nafion® 117, a common cation exchange membrane. Measurement errors for the permeation of mixtures of solutes are discussed. Permeabilities from one-, two-, and three-solute mixed aqueous solutions were measured using a standard diffusion cell, and ATR FTIR spectra were used to obtain time-resolved concentration data that were fit to a model describing transport of ions and small molecules through hydrated polymer films. The permeability of Nafion® 117 to methanol measured using this methodology was in agreement with literature reports. The sorption of methanol, sodium formate, and sodium acetate, and mixtures thereof, were measured using a desorption technique. From the measured permeabilities and solubilities, diffusivities of each solute were calculated. Differences in permeability among the solutes were found to be primarily due to differences in their solubility in Nafion® 117

Synthetic aptamer-polymer hybrid constructs for programmed drug delivery into specific target cells

Viruses have evolved specialized mechanisms to efficiently transport nucleic acids and other biomolecules into specific host cells. They achieve this by performing a coordinated series of complex functions, resulting in delivery that is far more efficient than existing synthetic delivery mechanisms. Inspired by these natural systems, we describe a process for synthesizing chemically defined molecular constructs that likewise achieve targeted delivery through a series of coordinated functions. We employ an efficient click chemistry technique to synthesize aptamer-polymer hybrids (APHs), coupling cell-targeting aptamers to block copolymers that secure a therapeutic payload in an inactive state. Upon recognizing the targeted cell-surface marker, the APH enters the host cell via endocytosis, at which point the payload is triggered to be released into the cytoplasm. After visualizing this process with coumarin dye, we demonstrate targeted killing of tumor cells with doxorubicin. Importantly, this process can be generalized to yield APHs that specifically target different surface markers.115850sciescopu