Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

Land use synergies and conflicts identification in the framework of compatibility analyses and spatial assessment of ecological, socio-cultural and economic values.

Land-use conflicts can be costly and time-consuming and cause social burden to all parties. In this study, we developed an approach for mapping synergy and conflict potential between land uses and tested it on nature protection, nature-based tourism, forestry and mining. First, we calculated the ecological and socio-cultural values for the study area, and further the economic values related to forestry and mining. Second, we conducted an integrated spatial assessment of these values and used it jointly with a variant of a value compatibility analysis to locate areas with possible synergistic and conflicting land uses. This study was carried out in Finnish Lapland where land use conflicts have occurred due to the need to develop forestry and mining in areas that are also important for nature-based tourism. The method operated well as it identified sites with ongoing land-use disputes. Synergy potential between biodiversity and socio-cultural values was identified in protected areas and other sites of natural beauty, and conflict potential concerning forestry near tourist resorts and concerning mining at proposed mining project sites. The developed framework can assist in locating sites that may need proactive measurements to avoid conflicts, and sites that would benefit from multi-purpose management thereby supporting sustainable and adaptive land-use planning.

The moderating role of the built environment in prenatal lifestyle interventions.

This study examined whether the neighborhood built environment moderated gestational weight gain (GWG) in LIFE-Moms clinical trials. Participants were 790 pregnant women (13.9 weeks' gestation) with overweight or obesity randomized within four clinical centers to standard care or lifestyle intervention to reduce GWG. Geographic information system (GIS) was used to map the neighborhood built environment. The intervention relative to standard care significantly reduced GWG (coefficient = 0.05; p = 0.005) and this effect remained significant (p < 0.03) after adjusting for built environment variables. An interaction was observed for presence of fast food restaurants (coefficient = -0.007; p = 0.003). Post hoc tests based on a median split showed that the intervention relative to standard care reduced GWG in participants living in neighborhoods with lower fast food density 0.08 [95% CI, 0.03,0.12] kg/week (p = 0.001) but not in those living in areas with higher fast food density (0.02 [-0.04, 0.08] kg/week; p = 0.55). Interaction effects suggested less intervention efficacy among women living in neighborhoods with more grocery/convenience stores (coefficient = -0.005; p = 0.0001), more walkability (coefficient -0.012; p = 0.007) and less crime (coefficient = 0.001; p = 0.007), but post-hoc tests were not significant. No intervention x environment interaction effects were observed for total number of eating establishments or tree canopy. Lifestyle interventions during pregnancy were effective across diverse physical environments. Living in environments with easy access to fast food restaurants may limit efficacy of prenatal lifestyle interventions, but future research is needed to replicate these findings.

Structural and functional characterization of the TYW3/Taw3 class of SAM-dependent methyltransferases.

S-adenosylmethionine (SAM)-dependent methyltransferases regulate a wide range of biological processes through the modification of proteins, nucleic acids, polysaccharides, as well as various metabolites. TYW3/Taw3 is a SAM-dependent methyltransferase responsible for the formation of a tRNA modification known as wybutosine and its derivatives that are required for accurate decoding in protein synthesis. Here, we report the crystal structure of Taw3, a homolog of TYW3 from Sulfolobus solfataricus, which revealed a novel α/ß fold. The sequence motif (S/T)xSSCxGR and invariant aspartate and histidine, conserved in TYW3/Taw3, cluster to form the catalytic center. These structural and sequence features indicate that TYW3/Taw3 proteins constitute a distinct class of SAM-dependent methyltransferases. Using site-directed mutagenesis along with in vivo complementation assays combined with mass spectrometry as well as ligand docking and cofactor binding assays, we have identified the active site of TYW3 and residues essential for cofactor binding and methyltransferase activity.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

Cane toads (Rhinella marina) rely on water access, not drought tolerance, to invade xeric Australian environments.

The invasion of habitats with novel environmental challenges may require physiological tolerances not seen in conspecifics from the native range. We used a combination of field and laboratory-based experiments to assess physiological tolerance to limited water access at four sites distributed across the historical invasion path of cane toads (Rhinella marina) in Australia that, from east to west, alternated between mesic and seasonally xeric habitats. Toads from all locations were well hydrated at the time of capture. However, experimental dehydration caused greater mass loss, higher plasma osmolality, and inhibition of lytic ability in toads from xeric compared to mesic locations. These results suggest somewhat surprisingly that toads from xeric environments are physiologically more vulnerable to water loss. In contrast, bactericidal ability was not sensitive to hydric state and was greater in toads from eastern (long-colonized) areas. Similar patterns in lytic ability in hydrated toads and agglutination ability in wild toads suggest that toads along the invasion front face a tradeoff between enhanced dispersal ability and physiological responses to dehydration. The ability of this invasive species to spread into drier environments may be underpinned by a combination of phenotypic plasticity and evolved (heritable) traits.

A family of metal-dependent phosphatases implicated in metabolite damage-control.

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg(2+) bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.

Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 Å resolution and revealed a modified α/ß hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies.

The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in kcat/Km Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.

Cardiac iron load and function in transfused patients treated with deferasirox (the MILE study).

OBJECTIVES: To assess the effect of iron chelation therapy with deferasirox on cardiac iron and function in patients with transfusion-dependent thalassemia major, sickle cell disease (SCD), and myelodysplastic syndromes (MDS). METHODS: This phase IV, single-arm, open-label study over 53 wk evaluated the change in cardiac and liver iron load with deferasirox (up to 40 mg/kg/d), measured by magnetic resonance imaging (MRI). RESULTS: Cardiac iron load (myocardial T2*) significantly improved (P = 0.002) overall (n = 46; n = 36 thalassemia major, n = 4 SCD, n = 6 MDS). Results were significant for patients with normal and moderate baseline cardiac iron (P = 0.017 and P = 0.015, respectively), but not in the five patients with severe cardiac iron load. Liver iron concentration (LIC) significantly decreased overall [mean LIC 10.4 to 8.2 mg Fe/g dry tissue (dw); P = 0.024], particularly in those with baseline LIC >7 mg Fe/g dw (19.9 to 15.6 mg Fe/g dw; P = 0.002). Furthermore, myocardial T2* significantly increased in patients with LIC <7 mg Fe/g dw, but not in those with a higher LIC. Safety was consistent with previous reports. CONCLUSIONS: Once-daily deferasirox over 1 yr significantly increased myocardial T2* and reduced LIC. This confirms that single-agent deferasirox is effective in the management of cardiac iron, especially for patients with myocardial T2* >10 ms (Clinicaltrials.gov identifier: NCT00673608).

Understanding the effects of different social data on selecting priority conservation areas.

Conservation success is contingent on assessing social and environmental factors so that cost-effective implementation of strategies and actions can be placed in a broad social-ecological context. Until now, the focus has been on how to include spatially explicit social data in conservation planning, whereas the value of different kinds of social data has received limited attention. In a regional systematic conservation planning case study in Australia, we examined the spatial concurrence of a range of spatially explicit social values and land-use preferences collected using a public participation geographic information system and biological data. We used Zonation to integrate the social data with the biological data in a series of spatial-prioritization scenarios to determine the effect of the different types of social data on spatial prioritization compared with biological data alone. The type of social data (i.e., conservation opportunities or constraints) significantly affected spatial prioritization outcomes. The integration of social values and land-use preferences under different scenarios was highly variable and generated spatial prioritizations 1.2-51% different from those based on biological data alone. The inclusion of conservation-compatible values and preferences added relatively few new areas to conservation priorities, whereas including noncompatible economic values and development preferences as costs significantly changed conservation priority areas (48.2% and 47.4%, respectively). Based on our results, a multifaceted conservation prioritization approach that combines spatially explicit social data with biological data can help conservation planners identify the type of social data to collect for more effective and feasible conservation actions.

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.

Identifying conflict potential in a coastal and marine environment using participatory mapping.

Planning for coastal and marine environments is often characterized by conflict over current and proposed uses. Marine spatial planning has been proposed as a way forward, however, social data are often missing impeding decision-making. Participatory mapping, a technique useful for providing social data and predict conflict potential, is being used in an increasing number of terrestrial applications to inform planning, but has been little used in the marine realm. This study collected social data for an extensive coastline in northwestern Australia via 167 in-depth face-to-face interviews including participant mapping of place values. From the transcribed interviews and digitized maps, we inductively identified 17 values, with biodiversity, the physical landscape, and Aboriginal culture being most valued. To spatially identify conflict potential, values were classified in matrices as consumptive or non-consumptive with the former assumed to be less compatible with other values. Pairwise comparisons of value compatibilities informed a spatial GIS determination of conflict potential. The results were overlaid with the boundaries of nine marine protected areas in the region to illustrate the application of this method for marine spatial planning. The three near shore marine protected areas had at least one third of their area exhibiting conflict potential. Participatory mapping accompanied by conflict potential mapping provides important insights for spatial planning in these often-highly contested marine environments.

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members.

Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/ß-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/ß-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

Emergency transport of stroke suspects in a rural state: opportunities for improvement.

INTRODUCTION: Time delay is the key obstacle for receiving successful stroke treatment. Alteplase therapy must start within 4.5 hours from stroke occurrence. Rapid transport to a primary stroke center (PSC) or acute stroke-ready hospital (ASRH) by the emergency medical system (EMS) paramedics is vital. We determined transport time and destination data for EMS-identified and -delivered stroke suspects in Arkansas during 2013. Our objective was to analyze transport time and the hospital qualification for stroke care across the state. METHODS: The state's 75 counties were placed into 8 geographical regions (R1-R8). Transport time and hospital qualification were determined for all EMS-identified strokes. Each hospital's stroke care status was categorized as PSC, ASRH, a nonspecialty or unknown care facility (NSCF), out-of-state, or nonapplicable designation facilities. RESULTS: There were 9588 EMS stroke ground transports with median within-region transport times of 29-40 minutes. Statewide, only 65% of EMS-transported stroke patients were transported to either PSC (12%) or ASRH (53%) facilities. One-third of the patients (30.6%) were delivered to NSCFs, where acute stroke therapy may rarely be performed. Regions with the highest suspected-stroke cases per capita also had the highest percentage of transports to NSCFs. CONCLUSION: With only a few PSCs in Arkansas, EMS agencies should prioritize transporting stroke patients to ASRHs when PSCs are not regionally located.

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.

Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

A Comparison of the Efficacy and Adverse Effects of Double-Lumen Endobronchial Tubes and Bronchial Blockers in Thoracic Surgery: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

OBJECTIVE: To compare the efficacy and adverse effects of using bronchial blockers (BBs) and double-lumen endobronchial tubes (DLTs). DESIGN: Systematic review and meta-analysis of randomized controlled trials (RCTs) comparing BBs and DLTs. SETTING: Hospital units undertaking thoracic surgery PARTICIPANTS: Patients undergoing thoracic surgery requiring lung isolation. INTERVENTIONS: BBs and DLTs. MEASUREMENTS AND MAIN RESULTS: A systematic literature search was conducted for RCTs comparing BBs and DLTs using Google Scholar, Ovid Medline, and Cochrane library databases up to October 2013. Inclusion criteria were RCTs comparing BBs and DLTs, intubation carried out by qualified anesthesiologists or trainee specialists, outcome measures relating to either efficacy or adverse effects. Studies that were inaccessible in English were excluded. Mantel-Haenszel fixed-effect meta-analysis of recurring outcome measures was performed using RevMan 5 software. The search produced 39 RCTs published between 1996 and 2013. DLTs were quicker to place (mean difference: 51 seconds, 95% confidence intervals [CI] 8-94 seconds; p = 0.02) and less likely to be incorrectly positioned (odds ratio [OR] 2.70; 95% CI 1.18-6.18, p = 0.02) than BBs. BBs were associated with fewer patients having a postoperative sore throat (OR 0.39, 95% CI: 0.23-0.68, p = 0.0009), less hoarseness (OR: 0.43,95%, CI 0.24-0.75, p = 0.003), and fewer airway injuries (OR 0.40, 95% CI 0.21-0.75, p = 0.005) than DLTs. CONCLUSION: While BBs are associated with a lower incidence of airway injury and a lower severity of injury, DLTs can be placed quicker and more reliably.