An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157: H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC(H7) mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC(H7) mutant O157 strain with fliC(H7) restored the adherence to wild-type levels; however, complementation with fliC(H6) did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

Roles of proteolysis in regulation of G protein-coupled receptor function

The enzymatic activity of peptidases must be tightly regulated to prevent uncontrolled hydrolysis of peptide bonds, which could have devastating effects in biological systems. Peptidases are often generated as inactive propeptidases, secreted with endogenous inhibitors or they are compartmentalized. Propeptidases become active after proteolytic removal of N-terminal activation peptides by other peptidases. Some peptidases only become active towards substrates only at certain pHs, thus confining activity to specific compartments or conditions. This review discusses the different roles proteolysis plays in regulating G protein-coupled receptors (GPCRs). At the cell-surface, certain GPCRs are regulated by the hydrolytic inactivation of bioactive peptides by membrane-anchored peptidases, which prevents signaling. Conversely, cell-surface peptidases can also generate bioactive peptides that directly activate GPCRs. Alternatively, cell-surface peptidases activated by GPCRs, can generate bioactive peptides to cause transactivation of receptor tyrosine kinases, thereby promoting signaling. Certain peptidases can signals directly to cells, by cleaving GPCR to initiate intracellular signaling cascades. Intracellular peptidases also regulate GPCRs; lysosomal peptidases destroy GPCRs in lysosomes to permanently terminate signaling and mediate downregulation; endosomal peptidases cleave internalized peptide agonists to regulate GPCR recycling, resensitization and signaling; and soluble intracellular peptidases also participate in GPCR function by regulating the ubiquitination state of GPCRs, thereby altering GPCR signaling and fate. Although the use of peptidase inhibitors has already brought success in the treatment of diseases such as hypertension, the discovery of new regulatory mechanisms involving proteolysis that control GPCRs may provide additional targets to modulate dysregulated GPCR signaling in disease.

Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

Sparse probability density function estimation using the minimum integrated square error

We develop a new sparse kernel density estimator using a forward constrained regression framework, within which the nonnegative and summing-to-unity constraints of the mixing weights can easily be satisfied. Our main contribution is to derive a recursive algorithm to select significant kernels one at time based on the minimum integrated square error (MISE) criterion for both the selection of kernels and the estimation of mixing weights. The proposed approach is simple to implement and the associated computational cost is very low. Specifically, the complexity of our algorithm is in the order of the number of training data N, which is much lower than the order of N2 offered by the best existing sparse kernel density estimators. Numerical examples are employed to demonstrate that the proposed approach is effective in constructing sparse kernel density estimators with comparable accuracy to those of the classical Parzen window estimate and other existing sparse kernel density estimators.

Apheresis donors and platelet function: inherent platelet responsiveness influences platelet quality

BACKGROUND: Process-induced platelet (PLT) activation occurs with all production methods, including apheresis. Recent studies have highlighted the range and consistence of interindividual variation in the PLT response, but little is known about the contribution of a donors' inherent PLT responsiveness to the activation state of the apheresis PLTs or the effect of frequent apheresis on donors' PLTs. STUDY DESIGN AND METHODS: The relationship between the donors' PLT response on the apheresis PLTs was studied in 47 individuals selected as having PLTs with inherently low, intermediate, or high responsiveness. Whole-blood flow cytometry was used to measure PLT activation (levels of bound fibrinogen) before donation and in the apheresis PLTs. The effects of regular apheresis on the activation status of donors' PLTs were studied by comparing the in vivo activation status of PLTs from apheresis (n = 349) and whole-blood donors (n = 157), before donation. The effect of apheresis per se on PLT activation was measured in 10 apheresis donors before and after donation. RESULTS: The level of PLT activation in the apheresis packs was generally higher than in the donor, and the most activated PLTs were from high-responder donors. There was no significant difference in PLT activation before donation between the apheresis and whole-blood donors (p = 0.697), and there was no consistent evidence of activation in the donors immediately after apheresis. CONCLUSION: The most activated apheresis PLTs were obtained from donors with more responsive PLTs. Regular apheresis, however, does not lead to PLT activation in the donors.

A fast algorithm for sparse probability density function construction

A new sparse kernel density estimator is introduced. Our main contribution is to develop a recursive algorithm for the selection of significant kernels one at time using the minimum integrated square error (MISE) criterion for both kernel selection. The proposed approach is simple to implement and the associated computational cost is very low. Numerical examples are employed to demonstrate that the proposed approach is effective in constructing sparse kernel density estimators with competitive accuracy to existing kernel density estimators.

Televised Whorf: cognitive restructuring in advanced foreign language learners as a function of audio-visual media exposure

The encoding of goal-oriented motion events varies across different languages. Speakers of languages without grammatical aspect (e.g., Swedish) tend to mention motion endpoints when describing events, e.g., “two nuns walk to a house,”, and attach importance to event endpoints when matching scenes from memory. Speakers of aspect languages (e.g., English), on the other hand, are more prone to direct attention to the ongoingness of motion events, which is reflected both in their event descriptions, e.g., “two nuns are walking.”, and in their non-verbal similarity judgements. This study examines to what extent native speakers of Swedish (n = 82) with English as a foreign language (FL) restructure their categorisation of goal-oriented motion as a function of their English proficiency and experience with the English language (e.g., exposure, learning). Seventeen monolingual native English speakers from the United Kingdom (UK) were engaged for comparison purposes. Data on motion event cognition were collected through a memory-based triads matching task, in which a target scene with an intermediate degree of endpoint orientation was matched with two alternative scenes with low and high degrees of endpoint orientation, respectively. Results showed that the preference among the Swedish speakers of L2 English to base their similarity judgements on ongoingness rather than event endpoints was correlated with their use of English in their everyday lives, such that those who often watched television in English approximated the ongoingness preference of the English native speakers. These findings suggest that event cognition patterns may be restructured through the exposure to FL audio-visual media. The results thus add to the emerging picture that learning a new language entails learning new ways of observing and reasoning about reality.

A radial basis function network classifier to maximise leave-one-out mutual information

tWe develop an orthogonal forward selection (OFS) approach to construct radial basis function (RBF)network classifiers for two-class problems. Our approach integrates several concepts in probabilisticmodelling, including cross validation, mutual information and Bayesian hyperparameter fitting. At eachstage of the OFS procedure, one model term is selected by maximising the leave-one-out mutual infor-mation (LOOMI) between the classifier’s predicted class labels and the true class labels. We derive theformula of LOOMI within the OFS framework so that the LOOMI can be evaluated efficiently for modelterm selection. Furthermore, a Bayesian procedure of hyperparameter fitting is also integrated into theeach stage of the OFS to infer the l2-norm based local regularisation parameter from the data. Since eachforward stage is effectively fitting of a one-variable model, this task is very fast. The classifier construc-tion procedure is automatically terminated without the need of using additional stopping criterion toyield very sparse RBF classifiers with excellent classification generalisation performance, which is par-ticular useful for the noisy data sets with highly overlapping class distribution. A number of benchmarkexamples are employed to demonstrate the effectiveness of our proposed approach.

Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

Location and characteristics of the reconnection X line deduced from low-altitude satellite and ground-based observations: 2. Defense Meteorological Satellite Program and European Incoherent Scatter data

We present an analysis of a cusp ion step, observed by the Defense Meteorological Satellite Program (DMSP) F10 spacecraft, between two poleward moving events of enhanced ionospheric electron temperature, observed by the European Incoherent Scatter (EISCAT) radar. From the ions detected by the satellite, the variation of the reconnection rate is computed for assumed distances along the open-closed field line separatrix from the satellite to the X line, do. Comparison with the onset times of the associated ionospheric events allows this distance to be estimated, but with an uncertainty due to the determination of the low-energy cutoff of the ion velocity distribution function, ƒ(ν). Nevertheless, the reconnection site is shown to be on the dayside magnetopause, consistent with the reconnection model of the cusp during southward interplanetary magnetic field (IMF). Analysis of the time series of distribution function at constant energies, ƒ(ts), shows that the best estimate of the distance do is 14.5±2 RE. This is consistent with various magnetopause observations of the signatures of reconnection for southward IMF. The ion precipitation is used to reconstruct the field-parallel part of the Cowley D ion distribution function injected into the open low-latitude boundary layer in the vicinity of the X line. From this reconstruction, the field-aligned component of the magnetosheath flow is found to be only −55±65 km s−1 near the X line, which means either that the reconnection X line is near the stagnation region at the nose of the magnetosphere, or that it is closely aligned with the magnetosheath flow streamline which is orthogonal to the magnetosheath field, or both. In addition, the sheath Alfvén speed at the X line is found to be 220±45 km s−1, and the speed with which newly opened field lines are ejected from the X line is 165±30 km s−1. We show that the inferred magnetic field, plasma density, and temperature of the sheath near the X line are consistent with a near-subsolar reconnection site and confirm that the magnetosheath field makes a large angle (>58°) with the X line.

The characteristics of the magnetopause reconnection X-line deduced from low-altitude satellite observations of cusp ions

We present an analysis of a “quasi-steady” cusp ion dispersion signature observed at low altitudes. We reconstruct the field-parallel part of the Cowley-D ion distribution function, injected into the open LLBL in the vicinity of the reconnection X-line. From this we find the field-parallel magnetosheath flow at the X-line was only 20 ± 60 km s−1, placing the reconnection site close to the flow streamline which is perpendicular to the magnetosheath field. Using interplanetary data and assuming the subsolar magnetopause is in pressure balance, we derive a wealth of information about the X-line, including: the density, flow, magnetic field and Alfvén speed of the magnetosheath; the magnetic shear across the X-line; the de-Hoffman Teller speed with which field lines emerge from the X-line; the magnetospheric field; and the ion transmission factor across the magnetopause. The results indicate that some heating takes place near the X-line as the ions cross the magnetopause, and that sheath densities may be reduced in a plasma depletion layer. We also compute the reconnection rate. Despite its quasi-steady appearance on an ion spectrogram, this cusp is found to reveal a large pulse of enhanced reconnection rate.

Elephants in pyjamas: testing the weak central coherence account of autism spectrum disorders using a syntactic disambiguation task

According to the weak central coherence (CC) account individuals with autism spectrum disorders (ASD) exhibit enhanced local processing and weak part-whole integration. CC was investigated in the verbal domain. Adolescents, recruited using a 2 (ASD status) by 2 (language impairment status) design, completed an aural forced choice comprehension task involving syntactically ambiguous sentences. Half the picture targets depicted the least plausible interpretation, resulting in longer RTs across groups. These were assumed to reflect local processing. There was no ASD by plausibility interaction and consequently little evidence for weak CC in the verbal domain when conceptualised as enhanced local processing. Furthermore, there was little evidence that the processing of syntactically ambiguous sentences differed as a function of ASD or language-impairment status.

Corticomuscular coherence modulation with the pattern of finger force coordination

We assess the corticomuscular coherence (CMC) of the contralateral primary motor cortex and the hand muscles during a finger force-tracking task and explore whether the pattern of finger coordination has an impact on the CMC level. Six healthy subjects (three men and three women) were recruited to conduct the force-tracking tasks comprising two finger patterns, i.e., natural combination of index and middle fingers and unnatural combination of index and middle fingers (i.e., simultaneously producing equal force strength in index and middle finger). During the conducting of the tasks with right index and middle finger, MEG and sEMG signals were recorded from left primary motor cortex (M1) and right flexor digitorum superficialis (FDS), respectively; the contralateral CMC was calculated to assess the neuromuscular interaction. Finger force-tracking tasks of Common-IM only induce beta-band CMC, whereas Uncommon-IM tasks produce CMC in both beta and low-gamma band. Compared to the force-tracking tasks of Common-IM, the Uncommon-IM task is associated with the most intensive contralateral CMC. Our study demonstrated that the pattern of finger coordination had significant impact on the CMC between the contralateral M1 and hand muscles, and more corticomuscular interaction was necessary for unnaturally coordinated finger activities to regulate the fixed neural drive of hand muscles.