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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.

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

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.

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]

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.

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.

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.

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