Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K(m) and V(max) values for UDP-GlcNAc, Mg(2+), and Mn(2+) suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg(2+), possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

Identification and molecular analysis of cable pilus biosynthesis genes in Burkholderia cepacia

Burkholderia cepacia is an opportunistic respiratory pathogen in cystic fibrosis patients. One highly transmissible and virulent clone belonging to genomovar IIIa expresses pili with unique cable morphology, which enable the bacterium to bind cytokeratin 13 in epithelial cells. The cblA gene, encoding the major pilin subunit, is often used as a DNA marker to identify potentially virulent isolates. The authors have now cloned and sequenced four additional genes, cblB, cblC, cblD and cblS, in the pilus gene cluster. This work shows that the products of the first four genes of the cbl operon, cblA, cblB, cblC and cblD, are sufficient for pilus assembly on the bacterial surface. Deletion of cblB abrogated pilus assembly and compromised the stability of the CblA protein in the periplasm. In contrast, deletion of cblD resulted in no pili, but there was no effect on expression and stability of the CblA protein subunit. These results, together with protein sequence homologies, predicted structural analyses, and the presence of typical amino acid motifs, are consistent with the assignment of functional roles for CblB as a chaperone that stabilizes the major pilin subunit in the periplasm, and CblD as the initiator of pilus biogenesis. It is also shown that expression of Cbl pili in Escherichia coli is not sufficient to mediate the binding of bacteria to the epithelial cell receptor cytokeratin 13, and that B. cepacia still binds to cytokeratin 13 in the absence of Cbl pili, suggesting that additional bacterial components are required for effective binding.

Allometric analysis of Sitka spruce branches:Mechanical versus hydraulic design principles

The geometry of tree branches can have considerable effect on their efficiency in terms of carbon export per unit carbon investment in structure. The purpose of this study was to evaluate different design criteria using data describing the form of Picea sitchensis branches. Allometric analysis of the data suggests that resources are distributed to favour shoots with the greatest opportunity for extension into new space, with priority to the extension of the leader. The distribution of allometric relations of links (branch elements) was tested against two models: the pipe model, based on hydraulic transport requirements, and a static load model based on the requirement of shoots to provide mechanical resistance to static loads. Static load resistance required the load parameter to be proportional to the link radius raised to the power of 4. This was shown to be true within a 95% statistical confidence limit. The pipe model would require total distal length to be proportional to link radius squared but the measured branches did not conform well to this model. The comparison suggests that the diameters of branch elements were more related to the requirements for mechanical load. The cost of following a hydraulic design principle (the pipe model) in terms of mechanical efficiency was estimated and suggested that the pipe model branch would not be mechanically compromised but would use structural resources inefficiently. Resource allocation among branch elements was found to be consistent with mechanical stability criteria but also indicated the possibility of allocation based on other criteria, such as potential light interception by shoots. The evidence suggests that whilst branch topology increments by reiteration of units of morphogenesis, the geometry follows a functional design pattern.

Cognitive, global, and functional benefits of donepezil in Alzheimer's disease and vascular dementia:results from large-scale clinical trials

Alzheimer's disease (AD) and vascular dementia (VaD) are both associated with deficits in cholinergic neurotransmission that are amenable to therapeutic intervention. The cholinesterase inhibitor, donepezil, is clinically effective in both AD and VaD. Results from a 10-study metaanalysis of donepezil (5 or 10 mg/day) in AD and a two-study combined analysis of donepezil (5 or 10 mg/day) in VaD are presented to compare patient characteristics and donepezil treatment outcomes. The analyzed studies were randomized, placebo-controlled, and of up to 24 weeks duration. In both AD and VaD, donepezil provided significant benefits compared with placebo on measures of cognition and global function. Placebo-treated AD patients showed a decline in cognition and global function, whereas placebo-treated VaD patients remained stable, suggesting treatment effects of donepezil in VaD were driven by improvement rather than stabilization or reduced decline. More VaD patients than AD patients received concomitant medications. Cardiovascular adverse events were more common in VaD than AD patients but were not increased by donepezil. In conclusion, although there are differences between AD and VaD patients in comorbid conditions and concomitant medications, donepezil is effective and well tolerated in both types of dementia.

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

Identifying functional stakeholder clusters to maximise communication for the ecosystem approach to fisheries management

Interaction with ecological models can improve stakeholder participation in fisheries management. Problems exist in efficiently communicating outputs to stakeholders and an objective method of structuring stakeholder differences is lacking. This paper aims to inform the design of a multi-user communication interface for fisheries management by identifying functional stakeholder groups. Intuitive categorisation of stakeholders, derived from survey responses, is contrasted with an Evidence-Based method derived from analysis of stakeholder literature. Intuitive categorisation relies on interpretation and professional judgement when categorising stakeholders among conventional stakeholder groups. Evidence-Based categorisation quantitatively characterises each stakeholder with a vector of four management objective interest-strength values (Yield, Employment, Profit and Ecosystem Preservation). Survey respondents agreed little in forming intuitive groups and the groups were poorly defined and heterogeneous in interests. In contrast the Evidence-Based clusters were well defined and largely homogeneous, so more useful for identifying functional relations with model outputs. The categorisations lead to two different clusterings of stakeholders and suggest unhelpful stereotyping of stakeholders may occur with the Intuitive categorisation method. Stakeholder clusters based on literature-evidence show a high degree of common interests among clusters and is encouraging for those seeking to maximise dialogue and consensus forming. © 2013 Elsevier Ltd.

Macroscopic limit of time-dependent density-functional theory for adiabatic local approximations of the exchange-correlation kernel

Time-dependent density-functional theory is a rather accurate and efficient way to compute electronic excitations for finite systems. However, in the macroscopic limit (systems of increasing size), for the usual adiabatic random-phase, local-density, or generalized-gradient approximations, one recovers the Kohn-Sham independent-particle picture, and thus the incorrect band gap. To clarify this trend, we investigate the macroscopic limit of the exchange-correlation kernel in such approximations by means of an algebraical analysis complemented with numerical studies of a one-dimensional tight-binding model. We link the failure to shift the Kohn-Sham spectrum of these approximate kernels to the fact that the corresponding operators in the transition space act only on a finite subspace.

Functional outcomes of decompressive hemicraniectomy following malignant middle cerebral artery infarctions: a systematic review

Decompressive hemicraniectomy has been used increasingly in recent years to treat malignant middle cerebral artery territory infarction. This review examines functional outcome data, with the novel analysis of outcomes according to temporal periods post-surgery. Case series data were pooled to determine significant correlates of outcome. Severe disability was frequently the outcome among survivors within one month post-surgery. Time and rehabilitation were later reflected, with fewer deaths and the emergence of mild to moderate disability increasing in prevalence. Mortality and severe disability were consistently more probable with increasing age. Presurgical clinical status in the form of additional cerebral artery involvement and midline shift also correlated with mortality within the 30-day period post-stroke.

Proteomic analysis of colorectal cancer metastasis:stathmin-1 revealed as a player in cancer cell migration and prognostic marker

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

The use of ensemble empirical mode decomposition with canonical correlation analysis as a novel artifact removal technique

Biosignal measurement and processing is increasingly being deployed in ambulatory situations particularly in connected health applications. Such an environment dramatically increases the likelihood of artifacts which can occlude features of interest and reduce the quality of information available in the signal. If multichannel recordings are available for a given signal source, then there are currently a considerable range of methods which can suppress or in some cases remove the distorting effect of such artifacts. There are, however, considerably fewer techniques available if only a single-channel measurement is available and yet single-channel measurements are important where minimal instrumentation complexity is required. This paper describes a novel artifact removal technique for use in such a context. The technique known as ensemble empirical mode decomposition with canonical correlation analysis (EEMD-CCA) is capable of operating on single-channel measurements. The EEMD technique is first used to decompose the single-channel signal into a multidimensional signal. The CCA technique is then employed to isolate the artifact components from the underlying signal using second-order statistics. The new technique is tested against the currently available wavelet denoising and EEMD-ICA techniques using both electroencephalography and functional near-infrared spectroscopy data and is shown to produce significantly improved results. © 1964-2012 IEEE.

Predicting Functional Recovery after Acute Ankle Sprain

INTRODUCTION:Ankle sprains are among the most common acute musculoskeletal conditions presenting to primary care. Their clinical course is variable but there are limited recommendations on prognostic factors. Our primary aim was to identify clinical predictors of short and medium term functional recovery after ankle sprain.
METHODS:A secondary analysis of data from adult participants (N = 85) with an acute ankle sprain, enrolled in a randomized controlled trial was undertaken. The predictive value of variables (age, BMI, gender, injury mechanism, previous injury, weight-bearing status, medial joint line pain, pain during weight-bearing dorsiflexion and lateral hop test) recorded at baseline and at 4 weeks post injury were investigated for their prognostic ability. Recovery was determined from measures of subjective ankle function at short (4 weeks) and medium term (4 months) follow ups. Multivariate stepwise linear regression analyses were undertaken to evaluate the association between the aforementioned variables and functional recovery.
RESULTS:Greater age, greater injury grade and weight-bearing status at baseline were associated with lower function at 4 weeks post injury (p<0.01; adjusted R square=0.34). Greater age, weight-bearing status at baseline and non-inversion injury mechanisms were associated with lower function at 4 months (p<0.01; adjusted R square=0.20). Pain on medial palpation and pain on dorsiflexion at 4 weeks were the most valuable prognostic indicators of function at 4 months (p< 0.01; adjusted R square=0.49).
CONCLUSION:The results of the present study provide further evidence that ankle sprains have a variable clinical course. Age, injury grade, mechanism and weight-bearing status at baseline provide some prognostic information for short and medium term recovery. Clinical assessment variables at 4 weeks were the strongest predictors of recovery, explaining 50% of the variance in ankle function at 4 months. Further prospective research is required to highlight the factors that best inform the expected convalescent period, and risk of recurrence.

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFN?(+) cells dominate disease initiation with IL-17(+) cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFN? responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFN? driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFN? cytokine production by CD4 and CD8 cells, but not IL-17 responses.

Advancing impact prediction and hypothesis testing in invasion ecology using a comparative functional response approach

Invasion ecology urgently requires predictive methodologies that can forecast the ecological impacts of existing, emerging and potential invasive species. We argue that many ecologically damaging invaders are characterised by their more efficient use of resources. Consequently, comparison of the classical ‘functional response’ (relationship between resource use and availability) between invasive and trophically analogous native species may allow prediction of invader ecological impact. We review the utility of species trait comparisons and the history and context of the use of functional responses in invasion ecology, then present our framework for the use of comparative functional responses. We show that functional response analyses, by describing the resource use of species over a range of resource availabilities, avoids many pitfalls of ‘snapshot’ assessments of resource use. Our framework demonstrates how comparisons of invader and native functional responses, within and between Type II and III functional responses, allow testing of the likely population-level outcomes of invasions for affected species. Furthermore, we describe how recent studies support the predictive capacity of this method; for example, the invasive ‘bloody red shrimp’ Hemimysis anomala shows higher Type II functional responses than native mysids and this corroborates, and could have predicted, actual invader impacts in the field. The comparative functional response method can also be used to examine differences in the impact of two or more invaders, two or more populations of the same invader, and the abiotic (e.g. temperature) and biotic (e.g. parasitism) context-dependencies of invader impacts. Our framework may also address the previous lack of rigour in testing major hypotheses in invasion ecology, such as the ‘enemy release’ and ‘biotic resistance’ hypotheses, as our approach explicitly considers demographic consequences for impacted resources, such as native and invasive prey species. We also identify potential challenges in the application of comparative functional responses in invasion ecology. These include incorporation of numerical responses, multiple predator effects and trait-mediated indirect interactions, replacement versus non-replacement study designs and the inclusion of functional responses in risk assessment frameworks. In future, the generation of sufficient case studies for a meta-analysis could test the overall hypothesis that comparative functional responses can indeed predict invasive species impacts.

Taxonomic and functional diversity of farmland bird communities across Europe:Effects of biogeography and agricultural intensification

In eight European study sites (in Spain, Ireland, Netherlands, Germany, Poland, Estonia and Sweden), abundance of breeding farmland bird territories was obtained from 500 × 500 m survey plots (30 per area, N = 240) using the mapping method. Two analyses were performed: (I) a Canonical Correspondence Analysis of species abundance in relation to geographical location and variables measuring agricultural intensification at field and farm level to identify significant intensification variables and to estimate the fractions of total variance in bird abundance explained by geography and agricultural intensification; (II) several taxonomic and functional community indices were built and analysed using GLM in relation to the intensification variables found significant in the CCA. The geographical location of study sites alone explains nearly one fifth (19. 5%) of total variation in species abundance. The fraction of variance explained by agricultural intensification alone is much smaller (4. 3%), although significant. The intersection explains nearly two fifths (37. 8%) of variance in species abundance. Community indices are negatively affected by correlates of intensification like farm size and yield, whereas correlates of habitat availability and quality have positive effects on taxonomic and functional diversity of assemblages. Most of the purely geographical variation in farmland bird assemblage composition is associated to Mediterranean steppe species, reflecting the bio-geographical singularity of that assemblage and reinforcing the need to preserve this community. Taxonomic and functional diversity of farmland bird communities are negatively affected by agricultural intensification and positively affected by increasing farmland habitat availability and quality. © 2011 Springer Science+Business Media B.V.

A generalized functional response for predators that switch between multiple prey species

We develop a theory for the food intake of a predator that can switch between multiple prey species. The theory addresses empirical observations of prey switching and is based on the behavioural assumption that a predator tends to continue feeding on prey that are similar to the prey it has consumed last, in terms of, e.g., their morphology, defences, location, habitat choice, or behaviour. From a predator's dietary history and the assumed similarity relationship among prey species, we derive a general closed-form multi-species functional response for describing predators switching between multiple prey species. Our theory includes the Holling type II functional response as a special case and makes consistent predictions when populations of equivalent prey are aggregated or split. An analysis of the derived functional response enables us to highlight the following five main findings. (1) Prey switching leads to an approximate power-law relationship between ratios of prey abundance and prey intake, consistent with experimental data. (2) In agreement with empirical observations, the theory predicts an upper limit of 2 for the exponent of such power laws. (3) Our theory predicts deviations from power-law switching at very low and very high prey-abundance ratios. (4) The theory can predict the diet composition of a predator feeding on multiple prey species from diet observations for predators feeding only on pairs of prey species. (5) Predators foraging on more prey species will show less pronounced prey switching than predators foraging on fewer prey species, thus providing a natural explanation for the known difficulties of observing prey switching in the field. (C) 2013 Elsevier Ltd. All rights reserved.