Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110:identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.

Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli

Lipopolysaccharide (LPS), a glycolipid molecule found on the outer leaflet of outer membranes of gram-negative bacteria, consists of three moieties: lipid A, core oligosaccharide, and the O-specific polysaccharide chain. The O-specific side chain, which extends to the extracellular milieu, plays an important role in pathogenicity, especially during the initial stages of infection, because of its ability to interact with serum complement. In recent years, several laboratories have used recombinant DNA tools to determine, at the molecular level, the organization, expression, and regulation of genes involved in LPS biosynthesis in Salmonella and Escherichia coli. An increased understanding of the molecular aspects of the O-specific side-chain genes will shed light on the intimate details related with the formation of the O-specific side chain, its assembly onto the lipid A--core, and the translocation and insertion of the complete LPS molecule into the outer membrane. It will also contribute to the understanding of the evolution of these genes and the correlation of chemical diversity of O-specific side chains with the genetic diversity of O-specific side-chain genes. In addition, since the O-specific side chains are involved in the pathogenicity of medically important gram-negative bacteria, a basic understanding of the regulation and expression of O-specific side chain LPS genes will contribute to the field of molecular pathogenesis. This article provides an overview of the role of O-specific side chains in septicemic infections and also discusses the current status of molecular genetic studies on O-specific side-chain genes from E. coli.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

From imagery to intention: A dual route model of imagined contact effects

Imagined intergroup contact (Crisp & R. Turner, 2009) is a new indirect contact strategy for promoting tolerance and more positive intergroup relations. In this chapter, we review existing research on imagined contact and propose two routes-cognitive and affective-through which it can exert a positive influence on contact-related attitudes and intentions. We first review research that has established the beneficial impacts of imagined contact on intergroup attitudes via reduced intergroup anxiety, supporting its efficacy as an intervention where there exists little or no opportunity for direct contact. We then review more recent research showing that imagined contact not only improves attitudes, but can also enhance intentions to engage in future contact. These studies suggest that contact imagery provides a behavioural script that forms the cognitive basis for subsequent judgements about future contact intentions. Collectively, the findings from this research programme support the idea that imagined contact can complement more direct forms of contact-providing a way of initially encouraging an interest in engaging positively with outgroups before introducing face-to-face encounters. We discuss the implications of these findings for future theory and research, and how they can inform prejudice-reduction interventions seeking to capitalise on the beneficial effects of mental imagery.

ARMS2 Increases the Risk of Early and Late Age-Related Macular Degeneration in the European Eye Study

OBJECTIVE:
To study associations between severity stages of early and late age-related macular degeneration (AMD) and genetic variations in age-related maculopathy susceptibility 2 (ARMS2) and complement factor H (CFH) and to investigate potential interactions between smoking and ARMS2.
DESIGN:
Population-based, cross-sectional European Eye Study in 7 countries in Europe.
PARTICIPANTS:
Four thousand seven hundred fifty participants, 65 years of age and older, recruited through random sampling.
METHODS:
Participants were classified on the basis of the more severely affected eye into 5 mutually exclusive AMD severity stages ranging from no AMD, 3 categories of early AMD, and late AMD. History of cigarette smoking was available and allowed classification into never, former, and current smokers, with the latter 2 groups combined into a single category of ever smokers for analysis. Genotyping was performed for single nucleotide polymorphisms rs10490924 and rs4146894 in ARMS2 and rs1061170 in CFH. Associations were analyzed by logistic regression.
MAIN OUTCOME MEASURES:
Odds ratios (ORs) for stage of AMD associated with genetic variations in ARMS2 and CFH and interactions between ARMS2 and smoking status.
RESULTS:
Early AMD was present in 36.4% and late AMD was present in 3.3% of participants. Data on both genotype and AMD were available for 4276 people. The ORs for associations between AMD stage and ARMS2 increased monotonically with more severe stages of early AMD and were altered little by adjustment for potential confounders. Compared with persons with no AMD, carriers of the TT genotype for rs10490924 in ARMS2 had a 10-fold increase in risk of late AMD (P<3 × 10(-20)). The ORs for associations with CFH were similar for stage 3 early AMD and late AMD. Interactions between rs10490924 in ARMS2 and smoking status were significant in both unadjusted and adjusted models (P = 0.001). The highest risk was observed in those doubly homozygous for rs10490924 and rs1061170 in CFH (OR, 62.3; 95% confidence interval, 16-242), with P values for trend ranging from 0.03 (early AMD, stage 1) to 1 × 10(-26) (late AMD).
CONCLUSIONS:
A strong association was demonstrated between all stages of AMD and genetic variation in ARMS2, and a significant gene-environment interaction with cigarette smoking was confirmed.

Cyclic behaviour of Volterra composition operators

We determine the cyclic behaviour of Volterra composition operators, which are defined as $(V_\phif)(x) =\int_0^{\phi(x)}f(t) dt$, $f ? L^p[0, 1]$, 1\leq p <\infty$,
where $?$ is a measurable self-map of [0, 1]. The cyclic behaviour of $V_\phi$ is essentially determined by the behaviour of the inducing symbol $\phi$ at 0 and at 1. As a particular result, we provide new examples of quasinilpotent supercyclic operators, which extend and complement previous ones of Hector Salas.

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days - 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) - to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg -1 furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 µg kg -1 minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.

Conjugated linoleic acid and atherosclerosis: no effect on molecular markers of cholesterol homeostasis in THP-1 macrophages

Macrophage cholesterol homeostasis is a key process involved in the initiation and progression of atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) regulate the transcription of the genes involved in cholesterol homeostasis and thus represent an important therapeutic target in terms of reducing atherosclerosis. Conjugated linoleic acid (CLA) is a potent anti-atherogenic dietary fatty acid in animal models of atherosclerosis and is capable of activating PPARs in vitro and in vivo. Therefore, this study examined whether the anti-atherogenic effects of CLA in vivo could be ascribed to altered cholesterol homeostasis in macrophages and macrophage derived foam cells. Of several genes that regulate cholesterol homeostasis investigated, CLA had most effect on the class B scavenger receptor CD36. The cis-9,trans-11 CLA (c9,t11-CLA) and trans-10,cis-12 CLA (t10,c12-CLA) isomers augmented CD36 mRNA expression (P

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux-marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. luxCDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at <10.26 microg ml(-1), bioluminescence was stimulated (e.g. 218% of control), but at concentrations >60 microg ml(-1) it declined to <1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5-25 microg ml(-1), approximately equals 55% of the added 14C was mineralized after 24 h compared with

The influence of evidence-based medicine training on decision-making in relation to over-the-counter medicines: a qualitative study

Objectives
To explore the role of evidence of effectiveness when making decisions about over-the-counter (OTC) medication and to ascertain whether evidence-based medicine training raised awareness in decision-making. Additionally, this work aimed to complement the findings of a previous study because all participants in this current study had received training in evidence-based medicine (unlike the previous participants).

Methods
Following ethical approval and an e-mailed invitation, face-to-face, semi-structured interviews were conducted with newly registered pharmacists (who had received training in evidence-based medicine as part of their MPharm degree) to discuss the role of evidence of effectiveness with OTC medicines. Interviews were recorded and transcribed verbatim. Following transcription, all data were entered into the NVivo software package (version 8). Data were coded and analysed using a constant comparison approach.

Key findings
Twenty-five pharmacists (7 males and 18 females; registered for less than 4 months) were recruited and all participated in the study. Their primary focus with OTC medicines was safety; sales of products (including those that lack evidence of effectiveness) were justified provided they did no harm. Meeting patient expectation was also an important consideration and often superseded evidence. Despite knowledge of the concept, and an awareness of ethical requirements, an evidence-based approach was not routinely implemented by these pharmacists. Pharmacists did not routinely utilize evidence-based resources when making decisions about OTC medicines and some felt uncomfortable discussing the evidence-base for OTC products with patients.

Conclusions
The evidence-based medicine training that these pharmacists received appeared to have limited influence on OTC decision-making. More work could be conducted to ensure that an evidence-based approach is routinely implemented in practice

COMPLETELY ITERATIVE, PIPELINED MULTIPLIER ARRAY SUITABLE FOR VLSI.

A pipelined array multiplier which has been derived by applying 'systolic array' principles at the bit level is described. Attention is focused on a circuit which is used to multiply streams of parallel unsigned data. Then an algorithm is given which demonstrates that, with only a simple modification to the basic cell, the same array can cope with two's complement numbers. The resulting structure has a number of features whch make it attractive to LSI and VLSI. These include regularity and modularity.

64-point Fourier transform chip for digital television applications

A 64-point Fourier transform chip is described that performs a forward or inverse, 64-point Fourier transform on complex two's complement data supplied at a rate of 13.5MHz and can operate at clock rates of up to 40MHz, under worst-case conditions. It uses a 0.6µm double-level metal CMOS technology, contains 535k transistors and uses an internal 3.3V power supply. It has an area of 7.8×8mm, dissipates 0.9W, has 48 pins and is housed in a 84 pin PLCC plastic package. The chip is based on a FFT architecture developed from first principles through a detailed investigation of the structure of the relevant DFT matrix and through mapping repetitive blocks within this matrix onto a regular silicon structure.

MULTIBIT CONVOLUTION USING A BIT LEVEL SYSTOLIC ARRAY.

A novel design for multibit convolver circuits is described. The circuits take the form of systolic arrays of simple one-bit processor and memory cells, with the result that they can operate at very high data rates and should be easy to implement using VLSI technology. An efficient method for handling two's complement data within the array is described and the relative advantages of this convolver design compared with more conventional circuits is discussed.

Programmable high-performance IIR filter chip

The paper presents a state-of-the-art commercial demonstrator chip for infinite impulse response (IIR) filtering. The programmable IIR filter chip contains eight multiplier/accumulators that can be configured in one of five different modes to implement up to a 16th-order IIR filter. The multiply-accumulate block is based on a highly regular systolic array architecture and uses a redundant number system to overcome problems of pipelining in the feedback loop. The chip has been designed using the GEC Plessey Semiconductors CLA 78000 series gate array, operates on 16-bit two's complement data and has a clock speed of 30 MHz. Issues such as overflow detection and design for testability have also been addressed and are described.