Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni

Aims: In Escherichia coli, increased expression of efflux pumps and/or decreased expression of porins can confer multiple antibiotic resistance (MAR), causing resistance to at least three unrelated classes of antibiotics, detergents and dyes. It was hypothesized that in Campylobacter jejuni, the efflux systems CmeABC, CmeDEF and the major outer membrane porin protein, MOMP (encoded by porA) could confer MAR. Methods: The expression of cmeB, cmeF and porA in 32 MAR C. jejuni isolated from humans or poultry was determined by comparative (C)-reverse transcriptase (RT)-PCR and denaturing DHPLC. A further 13 ethidium bromide-resistant isolates and three control strains were also investigated. Accumulation of ciprofloxacin carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was also determined for all strains. Results: Although resistance to ethidium bromide has been associated with MAR, expression of all three genes was similar in the ethidium bromide-resistant isolates. These data indicate that CmeB, CmeF and MOMP play no role in resistance to this agent in C. jejuni. Six MAR isolates over-expressed cmeB, 3/32 over-expressed cmeB and cmeF. No isolates over-expressed cmeF alone. Expression of porA was similar in all isolates. All nine isolates that over-expressed cmeB contained a mutation in cmeR, substituting glycine 86 with alanine. All cmeB over-expressing isolates also accumulated low concentrations of ciprofloxacin, which were restored to wild-type levels in the presence of CCCP. Conclusions: These data indicate that over-expression of cmeB is associated with MAR in isolates of C. jejuni. However, as cmeB was over-expressed by only one-third (9/32) of MAR isolates, these data also indicate other mechanisms of MAR in C. jejuni.

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli

Aims: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [ indicative of a multiple antibiotic resistance ( MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. Methods and Results: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli ( 10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive ( n = 5) and - resistant ( n = 5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli ( n = 389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations ( MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers ( Pless than or equal to 0(.)023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. Conclusions: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. Significance and Impact of the Study: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Chapter 17: X-Ray rheology of liquid crystal polymers

X-ray Rheology is an experimental technique which uses time-ressolved x-ray scattering as probe of the molecular level structural reorganisation which accompanies flow. It provides quantitative information on the direction alignment and on the level of global orientation. This information is very helpful in interpreting the classic rheological data on liquid crystal polymers. In this research we use data obtained from a cellulose derivate which exhibits a thermotropic liquid crystal phase. We show how increased shear rates lead to a rapid rise in the global orientation and we related this to therories of flow in liquid crystal polymers from the literature. We show that the relaxation time is independent of the prior shear rate.

On the solvability of second kind integral equations on the real line

This paper considers general second kind integral equations of the form(in operator form φ − kφ = ψ), where the functions k and ψ are assumed known, with ψ ∈ Y, the space of bounded continuous functions on R, and k such that the mapping s → k(s, · ), from R to L1(R), is bounded and continuous. The function φ ∈ Y is the solution to be determined. Conditions on a set W ⊂ BC(R, L1(R)) are obtained such that a generalised Fredholm alternative holds: If W satisfies these conditions and I − k is injective for all k ∈ W then I − k is also surjective for all k ∈ W and, moreover, the inverse operators (I − k) − 1 on Y are uniformly bounded for k ∈ W. The approximation of the kernel in the integral equation by a sequence (kn) converging in a weak sense to k is also considered and results on stability and convergence are obtained. These general theorems are used to establish results for two special classes of kernels: k(s, t) = κ(s − t)z(t) and k(s, t) = κ(s − t)λ(s − t, t), where κ ∈ L1(R), z ∈ L∞(R), and λ ∈ BC((R\{0}) × R). Kernels of both classes arise in problems of time harmonic wave scattering by unbounded surfaces. The general integral equation results are here applied to prove the existence of a solution for a boundary integral equation formulation of scattering by an infinite rough surface and to consider the stability and convergence of approximation of the rough surface problem by a sequence of diffraction grating problems of increasingly large period.

Interface vulnerability and knowledge of the subjunctive/indicative distinction with negated epistemic predicates in L2 Spanish.

Much recent research in SLA is guided by the hypothesis of L2 interface vulnerability (see Sorace 2005). This study contributes to this general project by examining the acquisition of two classes of subjunctive complement clauses in L2 Spanish: subjunctive complements of volitional predicates (purely syntactic) and subjunctive vs. indicative complements with negated epistemic matrix predicates, where the mood distinction is discourse dependent (thus involving the syntax-discourse interface). We provide an analysis of the volitional subjunctive in English and Spanish, suggesting that English learners of L2 Spanish need to access the functional projection Mood P and an uninterpretable modal feature on the Force head available to them from their formal English register grammar, and simultaneously must unacquire the structure of English for-to clauses. For negated epistemic predicates, our analysis maintains that they need to revalue the modal feature on the Force head from uninterpretable to interpretable, within the L2 grammar.With others (e.g. Borgonovo & Prévost 2003; Borgonovo, Bruhn de Garavito & Prévost 2005) and in line with Sorace's (2000, 2003, 2005) notion of interface vulnerability, we maintain that the latter case is more difficult for L2 learners, which is borne out in the data we present. However, the data also show that the indicative/subjunctive distinction with negated epistemics can be acquired by advanced stages of acquisition, questioning the notion of obligatory residual optionality for all properties which require the integration of syntactic and discourse information.

The structure of paranoia in the general population

Background: Psychotic phenomena appear to form a continuum with normal experience and beliefs, and may build on common emotional interpersonal concerns. Aims: We tested predictions that paranoid ideation is exponentially distributed and hierarchically arranged in the general population, and that persecutory ideas build on more common cognitions of mistrust, interpersonal sensitivity and ideas of reference. Method: Items were chosen from the Structured Clinical Interview for DSM-IV Axis II Disorders (SCID-II) questionnaire and the Psychosis Screening Questionnaire in the second British National Survey of Psychiatric Morbidity (n = 8580), to test a putative hierarchy of paranoid development using confirmatory factor analysis, latent class analysis and factor mixture modelling analysis. Results: Different types of paranoid ideation ranged in frequency from less than 2% to nearly 30%. Total scores on these items followed an almost perfect exponential distribution (r = 0.99). Our four a priori first-order factors were corroborated (interpersonal sensitivity; mistrust; ideas of reference; ideas of persecution). These mapped onto four classes of individual respondents: a rare, severe, persecutory class with high endorsement of all item factors, including persecutory ideation; a quasi-normal class with infrequent endorsement of interpersonal sensitivity, mistrust and ideas of reference, and no ideas of persecution; and two intermediate classes, characterised respectively by relatively high endorsement of items relating to mistrust and to ideas of reference. Conclusions: The paranoia continuum has implications for the aetiology, mechanisms and treatment of psychotic disorders, while confirming the lack of a clear distinction from normal experiences and processes.

Influences of increasing temperature on Indian wheat: quantifying limits to predictability

As climate changes, temperatures will play an increasing role in determining crop yield. Both climate model error and lack of constrained physiological thresholds limit the predictability of yield. We used a perturbed-parameter climate model ensemble with two methods of bias-correction as input to a regional-scale wheat simulation model over India to examine future yields. This model configuration accounted for uncertainty in climate, planting date, optimization, temperature-induced changes in development rate and reproduction. It also accounts for lethal temperatures, which have been somewhat neglected to date. Using uncertainty decomposition, we found that fractional uncertainty due to temperature-driven processes in the crop model was on average larger than climate model uncertainty (0.56 versus 0.44), and that the crop model uncertainty is dominated by crop development. Simulations with the raw compared to the bias-corrected climate data did not agree on the impact on future wheat yield, nor its geographical distribution. However the method of bias-correction was not an important source of uncertainty. We conclude that bias-correction of climate model data and improved constraints on especially crop development are critical for robust impact predictions.

Hydrology and geomorphology of the Upper White Nile lakes and their relevance for water resources management in the Nile basin

Remote sensing data and digital elevation models were utilized to extract the catchment hydrological parameters and to delineate storage areas for the Ugandan Equatorial Lakes region. Available rainfall/discharge data are integrated with these morphometric data to construct a hydrological model that simulates the water balance of the different interconnected basins and enables the impact of potential management options to be examined. The total annual discharges of the basins are generally very low (less than 7% of the total annual rainfall). The basin of the shallow (5 m deep) Lake Kioga makes only a minor hydrological contribution compared with other Equatorial Lakes, because most of the overflow from Lake Victoria basin into Lake Kioga is lost by evaporation and evapotranspiration. The discharge from Lake Kioga could be significantly increased by draining the swamps through dredging and deepening certain channel reaches. Development of hydropower dams on the Equatorial Lakes will have an adverse impact on the annual water discharge downstream, including the occasional reduction of flow required for filling up to designed storage capacities and permanently increasing the surface areas of water that is exposed to evaporation. On the basis of modelling studies, alternative sites are proposed for hydropower development and water storage schemes

Spatial patterns of gluten protein and polymer distribution in wheat grain

The starchy endosperm is the major storage tissue in the mature wheat grain and exhibits quantitative and qualitative gradients in composition, with the outermost cell layers being rich in protein, mainly gliadins, and the inner cells being low in protein but enriched in high-molecular-weight (HMW) subunits of glutenin. We have used sequential pearling to produce flour fractions enriched in particular cell layers to determine the protein gradients in four different cultivars grown at two nitrogen levels. The results show that the steepness of the protein gradient is determined by both genetic and nutritional factors, with three high-protein breadmaking cultivars being more responsive to the N treatment than a low-protein cultivar suitable for livestock feed. Nitrogen also affected the relative abundances of the three main classes of wheat prolamins: the sulfur-poor ω-gliadins showed the greatest response to nitrogen and increased evenly across the grain; the HMW subunits also increased in response to nitrogen but proportionally more in the outer layers of the starchy endosperm than near the core, while the sulfur-rich prolamins showed the opposite trend.

Present state of global wetland extent and wetland methane modelling: methodology of a model inter-comparison project (WETCHIMP)

The Wetland and Wetland CH4 Intercomparison of Models Project (WETCHIMP) was created to evaluate our present ability to simulate large-scale wetland characteristics and corresponding methane (CH4) emissions. A multi-model comparison is essential to evaluate the key uncertainties in the mechanisms and parameters leading to methane emissions. Ten modelling groups joined WETCHIMP to run eight global and two regional models with a common experimental protocol using the same climate and atmospheric carbon dioxide (CO2) forcing datasets. We reported the main conclusions from the intercomparison effort in a companion paper (Melton et al., 2013). Here we provide technical details for the six experiments, which included an equilibrium, a transient, and an optimized run plus three sensitivity experiments (temperature, precipitation, and atmospheric CO2 concentration). The diversity of approaches used by the models is summarized through a series of conceptual figures, and is used to evaluate the wide range of wetland extent and CH4 fluxes predicted by the models in the equilibrium run. We discuss relationships among the various approaches and patterns in consistencies of these model predictions. Within this group of models, there are three broad classes of methods used to estimate wetland extent: prescribed based on wetland distribution maps, prognostic relationships between hydrological states based on satellite observations, and explicit hydrological mass balances. A larger variety of approaches was used to estimate the net CH4 fluxes from wetland systems. Even though modelling of wetland extent and CH4 emissions has progressed significantly over recent decades, large uncertainties still exist when estimating CH4 emissions: there is little consensus on model structure or complexity due to knowledge gaps, different aims of the models, and the range of temporal and spatial resolutions of the models.

Granular flow in the marginal ice zone

The region of sea ice near the edge of the sea ice pack is known as the marginal ice zone (MIZ), and its dynamics are complicated by ocean wave interaction with the ice cover, strong gradients in the atmosphere and ocean and variations in sea ice rheology. This paper focuses on the role of sea ice rheology in determining the dynamics of the MIZ. Here, sea ice is treated as a granular material with a composite rheology describing collisional ice floe interaction and plastic interaction. The collisional component of sea ice rheology depends upon the granular temperature, a measure of the kinetic energy of flow fluctuations. A simplified model of the MIZ is introduced consisting of the along and across momentum balance of the sea ice and the balance equation of fluctuation kinetic energy. The steady solution of these equations is found to leading order using elementary methods. This reveals a concentrated region of rapid ice flow parallel to the ice edge, which is in accordance with field observations, and previously called the ice jet. Previous explanations of the ice jet relied upon the existence of ocean currents beneath the ice cover. We show that an ice jet results as a natural consequence of the granular nature of sea ice.

Computer simulations of pyroclastic flows from dome collapse

We present a method of simulating both the avalanche and surge components of pyroclastic flows generated by lava collapsing from a growing Pelean dome. This is used to successfully model the pyroclastic flows generated on 12 May 1996 by the Soufriere Hills volcano, Montserrat. In simulating the avalanche component we use a simple 3-fold parameterisation of flow acceleration for which we choose values using an inverse method. The surge component is simulated by a 1D hydraulic balance of sedimentation of clasts and entrainment of air away from the avalanche source. We show how multiple simulations based on uncertainty of the starting conditions and parameters, specifically location and size (mass flux), could be used to map hazard zones.

Terahertz pulse investigation of paleolithic wall etchings

Results are presented of an examination of flow rock-covered Paleoloithic cave art using time-domain terahertz reflectometry.