Sub-telomere directed gene expression during initiation of invasive aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

Comparative study of the hygienic behavior of Carniolan and Africanized honey bees directed towards grouped versus isolated dead brood cells

In Apis mellifera, hygienic behavior involves recognition and removal of sick, damaged or dead brood from capped cells. We investigated whether bees react in the same way to grouped versus isolated damaged capped brood cells. Three colonies of wild-type Africanized honey bees and three colonies of Carniolan honey bees were used for this investigation. Capped worker brood cells aged 12 to 14 days old were perforated with the pin-killing method. After making holes in the brood cells, the combs were placed back into the hives; 24 h later the number of cleaned cells was recorded in areas with pin-killed and control brood cells. Four repetitions were made in each colony. Isolated cells were more frequently cleaned than grouped cells, though variance analysis showed no significant difference (P = 0.1421). Carniolan bees also were somewhat, though not significantly more hygienic than Africanized honey bees (P = 0.0840). We conclude that honey bees can detect and remove both isolated and grouped dead brood. The tendency towards greater hygienic efficiency directed towards grouped pin-killed brood may be a consequence of a greater concentration of volatiles emanating from the wounds in the dead pupae.

System-Size Independence of Directed Flow Measured at the BNL Relativistic Heavy-Ion Collider

We measure directed flow (v(1)) for charged particles in Au + Au and Cu + Cu collisions at root s(NN) = 200 and 62.4 GeV, as a function of pseudorapidity (eta), transverse momentum (p(t)), and collision centrality, based on data from the STAR experiment. We find that the directed flow depends on the incident energy but, contrary to all available model implementations, not on the size of the colliding system at a given centrality. We extend the validity of the limiting fragmentation concept to v(1) in different collision systems, and investigate possible explanations for the observed sign change in v(1)(p(t)).

Directed flow at midrapidity in event-by-event hydrodynamics

Fluctuations in the initial geometry of a nucleus-nucleus collision have been recently shown to result in a new type of directed flow (v(1)) that, unlike the usual directed flow, is also present at midrapidity. We compute this new v(1) versus transverse momentum and centrality for Au-Au collisions at RHIC using the hydrodynamic code NeXSPheRIO. We find that the event plane of v(1) is correlated with the angle of the initial dipole of the distribution, as predicted, though with a large dispersion. It is uncorrelated with the reaction plane. Our results are in excellent agreement with results inferred from STAR correlation data.

Toxicity-directed approach of polyester manufacturing industry wastewater provides useful information for conducting treatability studies

A broader characterization of industrial wastewaters, especially in respect to hazardous compounds and their potential toxicity, is often necessary in order to determine the best practical treatment (or pretreatment) technology available to reduce the discharge of harmful pollutants to the environment or publicly owned treatment works. Using a toxicity-directed approach, this paper sets the base for a rational treatability study of polyester resin manufacturing. Relevant physical and chemical characteristics were determined. Respirometry was used for toxicity reduction evaluation after physical and chemical effluent fractionation. Of all the procedures investigated, only air stripping was significantly effective in reducing wastewater toxicity. Air stripping in pH 7 reduced toxicity in 18.2%, while in pH 11 a toxicity reduction of 62.5% was observed. Results indicated that toxicants responsible for the most significant fraction of the effluent`s instantaneous toxic effect to unadapted activated sludge were organic compounds poorly or not volatilized in acid conditions. These results led to useful directions for conducting treatability studies which will be grounded on actual effluent properties rather than empirical or based on the rare specific data on this kind of industrial wastewater. (C) 2008 Elsevier B.V. All rights reserved.

Galerkin finite element method for incompressible thermofluid flows framed within the bond graph theory

In this paper a bond graph methodology is used to model incompressible fluid flows with viscous and thermal effects. The distinctive characteristic of these flows is the role of pressure, which does not behave as a state variable but as a function that must act in such a way that the resulting velocity field has divergence zero. Velocity and entropy per unit volume are used as independent variables for a single-phase, single-component flow. Time-dependent nodal values and interpolation functions are introduced to represent the flow field, from which nodal vectors of velocity and entropy are defined as state variables. The system for momentum and continuity equations is coincident with the one obtained by using the Galerkin method for the weak formulation of the problem in finite elements. The integral incompressibility constraint is derived based on the integral conservation of mechanical energy. The weak formulation for thermal energy equation is modeled with true bond graph elements in terms of nodal vectors of temperature and entropy rates, resulting a Petrov-Galerkin method. The resulting bond graph shows the coupling between mechanical and thermal energy domains through the viscous dissipation term. All kind of boundary conditions are handled consistently and can be represented as generalized effort or flow sources. A procedure for causality assignment is derived for the resulting graph, satisfying the Second principle of Thermodynamics. (C) 2007 Elsevier B.V. All rights reserved.

Frequency Domain Connectivity Identification: An Application of Partial Directed Coherence in fMRI

Functional magnetic resonance imaging (fMRI) has become an important tool in Neuroscience due to its noninvasive and high spatial resolution properties compared to other methods like PET or EEG. Characterization of the neural connectivity has been the aim of several cognitive researches, as the interactions among cortical areas lie at the heart of many brain dysfunctions and mental disorders. Several methods like correlation analysis, structural equation modeling, and dynamic causal models have been proposed to quantify connectivity strength. An important concept related to connectivity modeling is Granger causality, which is one of the most popular definitions for the measure of directional dependence between time series. In this article, we propose the application of the partial directed coherence (PDC) for the connectivity analysis of multisubject fMRI data using multivariate bootstrap. PDC is a frequency domain counterpart of Granger causality and has become a very prominent tool in EEG studies. The achieved frequency decomposition of connectivity is useful in separating interactions from neural modules from those originating in scanner noise, breath, and heart beating. Real fMRI dataset of six subjects executing a language processing protocol was used for the analysis of connectivity. Hum Brain Mapp 30:452-461, 2009. (C) 2007 Wiley-Liss, Inc.

Optimization and Complexity Reduction of Switch-Reconfigured Antennas Using Graph Models

This letter addresses the optimization and complexity reduction of switch-reconfigured antennas. A new optimization technique based on graph models is investigated. This technique is used to minimize the redundancy in a reconfigurable antenna structure and reduce its complexity. A graph modeling rule for switch-reconfigured antennas is proposed, and examples are presented.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

The metal directed assembly of a trinuclear macrocyclic copper(II) complex

A trinuclear macrocyclic complex is reported from the metal directed condensation between melamine, formaldehyde and the Cu-II complex of a linear tetraamine.

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Tev sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. Ln support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the Rm-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork. (C) 1999 Academic Press.

Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: Site-directed mutagenesis and kinetic studies

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.

Active site-directed protein regulation

Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effecters bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.

Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

Metal-directed synthesis routes to a 16-membered tetraazamacrocycle with two pendant primary amine groups

The reaction of the bis(propane-1,3-diamine)copper(II) ion with paraformaldehyde and nitroethane in dry methanol under basic conditions produces a macrocyclic product, (cis-3,11-dimethyl-3,11-dinitro-1,5,9,13-tetraazacyclohexadecane)copper(II) perchlorate, in low yield, compared with the good yield obtained in the parallel chemistry possible even under aqueous conditions using palladium(II) as a template. The palladium complex was reduced with zinc amalgam in dilute aqueous acid to yield the metal-free 16-membered macrocyclic hexaamine, in this case re-complexed and characterised by an X-ray crystal structure as the (cis-3,11-dimethyl-1,5,9,13-tetraazacyclohexadecane-3,11-diamine)copper(II) perchlorate. The copper ion is found in a tetragonally elongated and trigonally-distorted octahedral environment, with all six of the ligand nitrogens coordinated, the two primary amine pendant groups occupying cis sites. (C) 2000 Elsevier Science S.A. All rights reserved.