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The β-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A β-lactamases, the β-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A β-lactamase with a ruthenocene-conjugated penicillin-a 0.85 A resolution structure of E166A mutant complexed with the penilloate product, a 1.30 A resolution complex structure of the same mutant with the penicilloate product, and a 1.18 A resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A β-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A β-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. Database Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.

When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5 ' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels. National Institutes of Heath [R21AI120602, R21AI139956, R03AI113648, P20GM113123]; Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health; High-End Instrumentation Grant [S10RR029127]; Office of the Vice President for Research; Markey Cancer Center; NCI Center Core Support Grant [P30 CA177558] These studies were funded by grants from the National Institutes of Heath: R21AI120602 (to B. Stevenson), R21AI139956 (to B. Stevenson, C.A. Brissette, W.R. Zuckert, and P. Schlax), R03AI113648 (to B. Stevenson, J. Seshu, and J. Livny) and P20GM113123 (to C.A. Brissette); and by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (to P.E. Stewart, K. Tilly, A. Bestor, and P.A Rosa). The Orbitrap mass spectrometer was acquired by High-End Instrumentation Grant S10RR029127 (to H. Zhu). The University of Kentucky Flow Cytometry & Immune Function core facility is supported in part by the Office of the Vice President for Research, the Markey Cancer Center and an NCI Center Core Support Grant (P30 CA177558) to the University of Kentucky Markey Cancer Center.

Maintenance of periodontal health or transition to a periodontal lesion reflects the continuous and ongoing battle between the vast microbial ecology in the oral cavity and the array of resident and emigrating inflammatory/immune cells in the periodontium. This war clearly signifies many 'battlefronts' representing the interface of the mucosal-surface cells with the dynamic biofilms composed of commensal and potential pathogenic species, as well as more recent knowledge demonstrating active invasion of cells and tissues of the periodontium leading to skirmishes in connective tissue, the locality of bone and even in the local vasculature. Research in the discipline has uncovered a concerted effort of the microbiome, using an array of survival strategies, to interact with other bacteria and host cells. These strategies aid in colonization by 'ambushing, infiltrating and outflanking' host cells and molecules, responding to local environmental changes (including booby traps for host biomolecules), communicating within and between genera and species that provide MASINT (Measurement and Signature Intelligence) to enhance sustained survival, sabotage the host inflammatory and immune responses and by potentially adopting a 'Fabian strategy' with a war of attrition and resulting disease manifestations. Additionally, much has been learned regarding the ever-increasing complexity of the host-response armamentarium at both cellular and molecular levels that is addressed in this review. Knowledge regarding how these systems fully interact requires both new laboratory and clinical tools, as well as sophisticated modeling of the networks that help maintain homeostasis and are dysregulated in disease. Finally, the triggers resulting in a 'coup de main' by the microbiome (exacerbation of disease) and the characteristics of susceptible hosts that can result in 'pyrrhic victories' with collateral damage to host tissues, the hallmark of periodontitis, remains unclear. While much has been learned, substantial gaps in our understanding of the 'parameters of this war' remain elusive toward fulfilling the Sun Tzu adage: 'If you know the enemy and know yourself, you need not fear the result of a hundred battles.'

Summary We compared nerve blockade with and without the InfinitiTM needle guide in an ultrasound in-plane porcine simulation. We recruited 30 anaesthetists with varying blockade experience. Using the guide, the needle tip was more visible (for a median (IQR [range]) of 67 (56–100]) % of the time; and invisible for 2 (1–4 [0–19]) s) than when the guide was not used (respectively 23 (13–43 [0–80]) % and 25 (9–52 [1–198]) s; both p

Releases of hydrocarbon-based fuels at retail filling stations have the potential to affect soil and groundwater and are listed by the European Commission as a potential pressure on achievement of the Water Framework Directive9s water quality objectives. This paper presents an analysis of the environmental sensitivity of retail filling station locations across Europe, with regard to proximity to groundwater, surface water and ecological receptors. A geographic information system-based approach was used to map the location of nearly 86 000 retail filling station sites to zones of differing environmental sensitivity. The results indicate that the environmental sensitivity of retail filling stations is highly variable, and that only a relatively small proportion has the potential to cause impact on groundwater abstractions, surface water or to designated ecological receptors. Accordingly, a site-specific risk-based approach to the design and operation of retail filling stations provides the most proportionate and sustainable basis for their management. In addition, the research provides a tool for both single retail filling station operators and the oil industry as a whole to identify areas of higher environmental sensitivity, encouraging the focusing of investment in preventive measures where it is most needed.

Borrelia burgdorferi, the causative agent of Lyme disease, survives in nature through a cycle that alternates between ticks and vertebrates. To facilitate this defined lifestyle, B. burgdorferi has evolved a gene regulatory network that ensures transmission between those hosts, along with specific adaptations to niches within each host. Several regulatory proteins are known to be essential for the bacterium to complete these critical tasks, but interactions between regulators had not previously been investigated in detail, due to experimental uses of different strain backgrounds and growth conditions. To address that deficit in knowledge, the transcriptomic impacts of four critical regulatory proteins were examined in a uniform strain background. Pairs of mutants and their wild-type parent were grown simultaneously under a single, specific culture condition, permitting direct comparisons between the mutant strains. Transcriptomic analyses were strand-specific, and assayed both coding and noncoding RNAs. Intersection analyses identified regulatory overlaps between regulons, including transcripts involved in carbohydrate and polyamine metabolism. In addition, it was found that transcriptional units such as ospC and dbpBA, which were previously observed to be affected by alternative sigma factors, are transcribed by RNA polymerase using the housekeeping sigma factor, RpoD.

Bioremediation was investigated as a method of treating a mixture of Forties Crude Oil and Heavy Fuel Oil stranded on Bullwell Bay, Milford Haven, UK after the grounding of the Sea Empress in 1996. A randomised block design in triplicate was used to test the efficacy of two bioremediation treatments: a weekly application of mineral nutrients dissolved in seawater and a single application of a slow-release fertilizer. Each treatment supplied an equivalent amount of nitrogen and phosphorus. Concentrations of residual hydrocarbons normalised to the biomarker 17α(H),21β(H)-hopane showed that after two months the oil was significantly (p

Biomass of the biocontrol agent Idriella bolleyi can be produced in submerged liquid culture as mycelium, conidia, and chlamydospores. Each fraction was separated and quantified. The conidial fraction appeared to be the most thermotolerant, suggesting that this form had the best potential shelf life of the three. Conidia were produced during the early phase of growth and their production did not appear to require any form of nutrient exhaustion or growth rate decay. The highest viability per unit biomass was obtained in carbon-limited medium, with sodium nitrate as the nitrogen source, after 6 h. Maximum biomass was not obtained in this medium until 167 h and, as the biocontrol product is likely to be sold by weight, an acceptable compromise between the two parameters must be found. Excess ammonium chloride in the medium appeared to inhibit growth. A procedure, involving culture homogenization, was adopted in order to estimate potential viability in shake-flask liquid culture.

The kinetics of erythromycin production were dependent on the identity of the growth rate-limiting nutrient during batch cultures of Saccharopolyspora erythraea. Semilogarithmic linear regression provided a single estimate of growth rate during the exponential phase, but partial cubic spline curve fit derivatives provided time-dependent specific growth and production rate profile. Non-growth-linked product formation was observed when the medium was glucose- or phosphate-limited. However, growth-linked product formation was observed in a nitrate-limited medium. The kinetics observed in nitrate-limited chemostat culture provided evidence that Saccharopolyspora erythraea may be subject to noncompetitive inhibition by a growth-linked product under these conditions. A mathematical model was used to test this theory. The model simulation fitted the observed data very closely and was used to calculate estimates of the kinetic parameters involved: [formula; see text]

Coulomb explosion has been promoted in a range of doubly charged atomic and molecular clusters. In these new experiments, mass selected clusters of Ar2+n, (CO2)2+n, (H2O)2+n, (H2O)nH2+2, (CH3CN)nH2+2, and (C6H6)2+n have been subjected to collisional activation with a background gas. For species close to the Coulomb cutoff, each collision removes sufficient atoms or molecules (approximately six) as to render the clusters unstable. As a result, charge separation occurs and part (≂30%) of the Coulomb repulsion energy is released in the form of center of mass kinetic energy in the fragments. The remaining Coulomb energy appears as internal excitation in the fragments and subsequently leads to extensive evaporation. It is shown that the latter process is continuing even 10−6 s after Coulomb explosion. All the molecular systems studied show evidence of asymmetric charge separation, with some singly charged fragments containing up to 65% of the initial cluster mass. A detailed quantitative analysis of the result...

A series of hard‐sphere cluster‐ion structures have been constructed, based on the notion of a central stable dimer ion core. Starting from a structure representative of icosahedral Ar+19, two separate packing sequences are considered; edge packing and face packing, and clusters containing up to 310 atoms have been generated. It is shown that many of the symmetric structures derived from the two packing sequences coincide with the ‘‘magic’’ numbers observed both in the mass spectra and fragmentation patterns of argon and krypton cluster ions. A slice through the cluster shows that the central dimer ion core is part of a linear chain of atoms, which offer the possibility for further charge delocalization as suggested in recent molecular orbital calculations.

A digital Class-D amplifier based on single-bit processing to be implemented on a field programmable gate array (FPGA) is presented. The main focus of this design is the reduction of noise, a drawback of Class-D amplifiers, by exploring the noise-shaping characteristics of sigma-delta modulation (SDM). The audio signal is retained in the digital single-bit domain until the final stage of the amplifier, thus avoiding quantization noise due to conversion. This paper focuses on the SDM and digital signal processing (DSP) components of the design to be implemented on the FPGA.

A reliable, cheap, simple and rapid method for measuring petroleum hydrocarbon contamination of soil out in the field has been developed to aid with the investigation and remediation of sites where petroleum hydrocarbons have been handled such as: retail filling stations, distribution depots, refineries, oil and gas terminals and onshore oil and gas production sites. It is useful for identifying and delineating any zones of contamination in the soil and monitoring the progress of site clean-up. No experience of analytical chemistry is necessary to be able to use the method effectively.

Bioremediation is capable of reducing the petroleum hydrocarbon concentration of soil contaminated with crude oil by 75–95%, depending on the soil type, the nature of the crude and the extent of weathering that has taken place prior to starting the clean-up. However, this degree of cleaning may be insufficient to meet some of the more stringent clean-up targets set by some regulatory authorities. It is therefore necessary to understand the reasons for incomplete clean-up, to determine whether anything can realistically be done about it and, if not, to help assess any hazard that might be posed by leaving the hydrocarbon residue in the ground.

Knowledge of technology performance under different contamination scenarios is key to effective risk-based corrective action (RBCA). The combination of soil vapour extraction and bioremediation (a.k.a. ‘bioventing’) has been promoted as a cost-effective technology to remediate mixed gasoline and diesel spills. It is therefore worthwhile to systematise its RBCA potential. ‘Rules-of-thumb’ (heuristics) to perform a technology screening, design, operation and monitoring of bioventing are necessary. Variables such as contaminant characteristics, well design, positioning and operation; and performance monitoring can all have significant cost implications.

Experiments and calculations designed to probe the structure of inert-gas cluster ions are discussed. Examples are drawn from studies on the unimolecular fragmentation of large argon and krypton cluster ions. Preliminary results are presented from measurements of the kinetic-energy release associated with the unimolecular decay of argon cluster ions.

Dynamic Programming (or DP as it is commonly known) is a mathematical programming method which would appear to be a very powerful technique for use in management decision problems. A great number of theoretical texts have been written on the mathematics of DP and a few articles have been published on the more practical aspects, but DP has remained very much on the theoretical shelf as far as practising management has been concerned. This paper gives the results of a survey carried out at the beginning of 1972 and is intended to provide some insight into the use of DP in real management problems in U.K. companies and to show what sort of problems are apparently restricting its use. The findings demonstrate that several firms have used DP in various applications with considerable success. There are also some enlightening comments on the difficulties involved, and on the future potential of DP in industry.

The ability of two cultivars of spring wheat, Cadet and Rescue, and their reciprocal chromosome substitution lines, C-R5D and R-C5D, to obtain significant quantities of N from atmospheric N2 was investigated in glasshouse experiments using 125N dilution. The wheat was inoculated with N2-fixing bacteria, including Azotohacter, Azospirillum, Klebsiella and Bacillus spp, in pure and mixed culture, at N concentrations ranging from 1 to 56 mg N plant−1 (14–168 μg N ml−1), in sand culture and in three soils of differing N content. Root-ussociutcd N2-fixalion was negligible unless carbohydrate was added to the rooting medium. Atmospheric N2 was incorporated into wheat roots and translocated to the tops, when plants inoculated with Azotobacter beijerinckii or Azospirillum brasilense sp. 107 were amended with glucose and malate respectively, under monoxenic conditions in sand culture.

Development of understanding of mathematical concepts and facility with quantitative techniques as aids to decision-making are important aims for post-experience management courses. Unfortunately, many British managers have a low level of numeracy and find this area difficult. From practical experience with such courses, it is argued that some of the special difficulties and the widely varying needs of middleaged managers might be met by introducing a self-scheduling learning scheme. The features of this approach to teaching are discussed against a background of the widespread interest in individualized learning in other areas of education. A proposed system for teaching quantitative techniques is described to illustrate this approach and raise a number of design issues.

A high resolution double focusing mass spectrometer in association with a pulsed supersonic nozzle has been used to study the unimolecular fragmentation of Ar+n clusters for n in the range 30–200. All ion clusters within this range are observed to undergo the reaction Ar+n→Ar+n−1+Ar; with some ions also exhibiting further fragmentation involving the loss of two and three argon atoms. A systematic study of the above reaction as a function of n reveals that the relative intensities of the fragment ions fluctuate considerably. In many cases, these fluctuations coincide with depleted or enhanced ion intensities in the normal ion cluster mass spectrum. Through the use of a model based on unimolecular reaction rate theory (RRKM), it has been possible to reproduce many of the features observed in both the fragmentation spectra and the normal mass spectrum. Of the two principle variables involved in the model, reaction symmetry number and dissociation or binding energy, we have been able to show that only the bin...

The effect of three organophosphorus insecticides on soil urease was examined. Inhibition of urea hydrolysis, some 60 days after application of 1000 parts/106 of insecticide to a sandy clay loam, approached 40% (accothion) and exceeded 50% in the case of malathion and thimet. Similar inhibitory effects were recorded using a silt loam soil with which 200 parts/106 application also produced inhibition ranging from 14% (accothion) to 23% (thimet) after 10 days. With lower concentrations of insecticide (50 parts/106) inhibition, though again significant, was of a more ephemeral nature. All three insecticides, at a concentration of 1000 parts/10+6, prevented almost any hydrolysis of urea by jack bean urease. Ureolytic microorganisms, isolated from the soils under investigation, were inhibited by the organophosphates to a greater or lesser extent but the development of tolerance was common. It is suggested that the application of insecticides to control soil-borne insect pests may be a factor in determining the efficiency of urea fertilizer mineralization.

A double‐focusing mass spectrometer in conjunction with a cluster beam source has been used to study the unimolecular fragmentation patterns of krypton cluster ions, Kr+n, for n in the range 10–340. All cluster ions are observed to undergo the reaction Kr+n →Kr+n−1 + Kr; however, some clusters lose up to five atoms on the time scale of the experiment. The six naturally occurring isotopes of krypton complicates the cluster‐ion mass spectra, with the number of ion peaks increasing rapidly with n for a given Kr+n. A computer model which successfully accounts for the influence of isotopes reveals that isotope enrichment of the cluster ions occurs. The relative intensities of the fragment ions exhibits pronounced intensity fluctuations, many of which coincide with similar observations on large argon cluster ions. It is proposed that particularly intense fragments, i.e., Kr+111 and Kr+120, arise from reactions which lead to the formation of kinetically favorable products. In contrast, intense ion peaks in the m...

A theoretical discussion is presented of the design features that have been incorporated into a multipass cell appropriate for use in laser molecular‐beam experiments. A detailed description of the cell in two dimensions is given, and this is later expanded to highlight the problems of alignment in three dimensions. A prototype cell, constructed using the guidelines presented here, has demonstrated that 31 passes through a molecular beam can be easily achieved. Computer simulations suggest that the optimum potential of the cell is ≊101 passes. The fact that the position of the emergent laser beam is clearly defined makes the cell particularly useful for the purposes of studying absorption spectra.

Etude de la dissociation unimoleculaire d'agregats ioniques de dioxyde de carbone, d'argon et d'eau. Pas de mise en evidence d'explosion coulombienne dans les systemes etudies

1.3-β-Glucanase (laminarinase) activity in soil was measured using laminarin as the substrate. Activity was optimal in sodium acid-maleate buffer at pH 5.4 and followed Michaelis-Menten kinetics. Three methods of analysing kinetic data gave Km values of 0.23, 0.21 and 0.20 mg.ml−1. Vmax values were 0.41, 0.39 and 0.39 μmole glucose, g−1. h−1. The activation energy of the reaction was 49 kJ. mole−1. A proportion of the activity was highly resistant to storage at various temperatures: at 50 C 1,3-β-glucanase had a half-life of 28 days.

Arylsulphatase, β-1,3 glucanase, phosphatase and urease responded differently to γ-irradiation (5–50 Mrad) in air-dried and moist soils. In all instances phosphatase was the most stable. The variability between enzymes may be due to inherent biochemical and stuctural characteristics or to their location within the soil microenvironment.

There is some overlap between our analysis and work previously published by Zipin, but the former is more applicable to the specific problem addressed.

Infrared absorption profiles have been measured for small acetonitrile clusters by monitoring the depletion of signal intensity in a mass spectrometer as a function of CO2 laser wavelength. The profiles have been compared with infrared spectra calculated for (CH3CN)4 using computer simulation methods. Classical mechanics has been used to treat the translational and rotational degrees of freedom, and perturbation theory has provided a measure of the frequencey shift induced in individual molecules by the surrounding environment. It is proposed that small acetronitrile clusters are solid-like with an internal temperature of