Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion.

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M. C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851-863). In this study, we report the cloning and initial characterization of a new paxillin LD motif-binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell-cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

Connectivity steered graph Fourier transform for motor imagery BCI decoding.

OBJECTIVE: Graph signal processing (GSP) concepts are exploited for brain activity decoding and particularly the detection and recognition of a motor imagery (MI) movement. A novel signal analytic technique that combines graph Fourier transform (GFT) with estimates of cross-frequency coupling (CFC) and discriminative learning is introduced as a means to recover the subject's intention from the multichannel signal. APPROACH: Adopting a multi-view perspective, based on the popular concept of co-existing and interacting brain rhythms, a multilayer network model is first built from empirical data and its connectivity graph is used to derive the GFT-basis. A personalized decoding scheme supporting a binary decision, either 'left versus right' or 'rest versus MI', is crafted from a small set of training trials. Electroencephalographic (EEG) activity from 12 volunteers recorded during two randomly alternating, externally cued, MI tasks (clenching either left or right fist) and a rest condition is used to introduce and validate our methodology. In addition, the introduced methodology was further validated based on dataset IVa of BCI III competition. MAIN RESULTS: Our GFT-domain decoding scheme achieves nearly optimal performance and proves superior to alternative techniques that are very popular in the field. SIGNIFICANCE: At a conceptual level, our work suggests a fruitful way to introduce network neuroscience in BCI research. At a more practical level, it is characterized by efficiency. Training is realized using a small number of exemplar trials and decoding requires very simple operations that leaves room for real-time implementation.

Discriminative codewaves: a symbolic dynamics approach to SSVEP recognition for asynchronous BCI.

OBJECTIVE: Steady-state visual evoked potential (SSVEP) is a very popular approach to establishing a communication pathway in brain-computer interfaces (BCIs), without any training requirements for the user. Brain activity recorded over occipital regions, in association with stimuli flickering at distinct frequencies, is used to predict the gaze direction. High performance is achieved when the analysis of multichannel signal is guided by the driving signals. This study introduces an efficient way of identifying the attended stimulus without the need to register the driving signals. APPROACH: Regional brain response is described as a dynamical trajectory towards one of the 'attractors' associated with the brainwave entrainment induced by the attended stimulus. A condensed description for each single-trial response is provided by means of discriminative vector quantization, and different trajectories are disentangled based on a simple classification scheme that uses templates and confidence intervals derived from a small training dataset. MAIN RESULTS: Experiments, based on two different datasets, provided evidence that the introduced approach compares favorably to well-established alternatives, regarding the information transfer rate. SIGNIFICANCE: Our approach relies on (but not restricted to) single sensor traces, incorporates a novel description of brainwaves based on semi-supervised learning, and its great advantage stems from its potential for self-paced BCI.

Preseismic electromagnetic signals in terms of complexity.

There is a recent thesis in the literature that an important organization of a physical system precedes a catastrophic event. In this context, one can search for signatures that imply the transition from a normal state to a main catastrophic event (e.g., earthquake). Experimental techniques are thus useful in corroborating theories from observed data. For example, recent results indicate that preseismic electromagnetic time series contain information characteristic of an ensuing earthquake event. Hereby, we attempt to demonstrate that an easily computable complexity measure, such as T-complexity or approximate entropy, gives evidence of state changes leading to the point of global instability. The appearance of a precatastrophic state is characterized by significant lower complexity in terms of T-complexity and approximate entropy. The present study confirms the conclusions of previous works based on an independent linear fractal spectral analysis. This convergence between nonlinear and linear analysis provides a more reliable detection concerning the emergence of the last phase of the earthquake preparation process. More precisely, we claim that our results suggest an important principle: significant complexity decrease and accession of persistency in electromagnetic (EM) time series can be confirmed at the tail of the preseismic EM emission, which could be used as diagnostic tools for the Earth's impending crust failure. Direct laboratory and field experimental data as well as theoretical arguments support the conclusions of the present analysis.

The human non-muscle alpha-actinin protein encoded by the ACTN4 gene suppresses tumorigenicity of human neuroblastoma cells.

alpha-Actinins are actin-binding proteins important in organization of the cytoskeleton and in cell adhesion. We have cloned and characterized a cDNA from human neuroblastoma cell variants which encodes the second non-muscle alpha-actinin isoform designated ACTN4 (actinin-4). mRNA encoded by the ACTN4 gene, mapped to chromosome 4, is abundant in non-tumorigenic, substrate-adherent human neuroblastoma cell variants but absent or only weakly expressed in malignant, poorly substrate-adherent neuroblasts. It is also present in many adherent tumor cell lines of diverse tissue origins. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle alpha-actinin gene. Expression is correlated with substrate adhesivity. Analysis of deduced amino acid sequences suggests that the two isoforms may differ in function and in regulation by calcium. Moreover, ACTN4 exhibits tumor suppressor activity. Stable clones containing increased levels of alpha-actinin, isolated from highly malignant neuroblastoma stem cells [BE(2)-C] after transfection with a full-length ACTN4 cDNA, show decreased anchorage-independent growth ability, loss of tumorigenicity in nude mice, and decreased expression of the N-myc proto-oncogene.

Isolation and characterization of the human secretogranin II gene promoter.

The goal of this study was to isolate and functionally characterize the human secretogranin II (SgII) gene promoter. SgII is a member of the granin family of proteins which are selectively expressed in neurosecretory cells. The human SgII promoter contains a consensus TATA box and cyclic AMP response element (CRE) 35 and 74 bp upstream of the transcription start site, respectively, elements also found in the mouse and rat SgII gene promoters. Transfection studies showed that 869 bp of the human SgII promoter were sufficient to confer cell type-specific expression of an SgII promoter-luciferase reporter gene in neurosecretory PC-12, GH and BE(2)-M17 cells. The activity of the human SgII promoter was also compared in three N-type, human neuroblastoma cell lines [BE(2)-M17, SMS-KAN and SH-SY5Y], which differ markedly in the level of SgII expression. SgII promoter activities in the neuroblastoma cell lines correlated not only with the levels of SgII but also the levels of the cyclic AMP response element-binding protein CREB which were highest in BE(2)-M17 cells and lowest in SH-SY5Y cells. To establish that the activity of the human SgII promoter in these neuroblastoma cell lines is dependent on the level of CREB, rat CREB was overexpressed in SH-SY5Y cells. SgII promoter activity was up to 8-fold higher in SH-SY5Y cells overexpressing CREB. These results suggest that SgII expression is a marker for neuronal differentiation in human neuroblastoma cell lines and is dependent on the level of CREB expression.

EPILAB: a software package for studies on the prediction of epileptic seizures.

A Matlab®-based software package, EPILAB, was developed for supporting researchers in performing studies on the prediction of epileptic seizures. It provides an intuitive and convenient graphical user interface. Fundamental concepts that are crucial for epileptic seizure prediction studies were implemented. This includes, for example, the development and statistical validation of prediction methodologies in long-term continuous recordings. Seizure prediction is usually based on electroencephalography (EEG) and electrocardiography (ECG) signals. EPILAB is able to process both EEG and ECG data stored in different formats. More than 35 time and frequency domain measures (features) can be extracted based on univariate and multivariate data analysis. These features can be post-processed and used for prediction purposes. The predictions may be conducted based on optimized thresholds or by applying classifications methods such as artificial neural networks, cellular neuronal networks, and support vector machines. EPILAB proved to be an efficient tool for seizure prediction, and aims to be a way to communicate, evaluate, and compare results and data among the seizure prediction community.

A computational environment for long-term multi-feature and multi-algorithm seizure prediction.

The daily life of epilepsy patients is constrained by the possibility of occurrence of seizures. Until now, seizures cannot be predicted with sufficient sensitivity and specificity. Most of the seizure prediction studies have been focused on a small number of patients, and frequently assuming unrealistic hypothesis. This paper adopts the view that for an appropriate development of reliable predictors one should consider long-term recordings and several features and algorithms integrated in one software tool. A computational environment, based on Matlab (®), is presented, aiming to be an innovative tool for seizure prediction. It results from the need of a powerful and flexible tool for long-term EEG/ECG analysis by multiple features and algorithms. After being extracted, features can be subjected to several reduction and selection methods, and then used for prediction. The predictions can be conducted based on optimized thresholds or by applying computational intelligence methods. One important aspect is the integrated evaluation of the seizure prediction characteristic of the developed predictors.

Low stimulation of peripheral lymphocytes, following in vitro application of Emdogain.

Fast tissue regeneration after therapeutic manipulations is a central problem of periodontology, oral surgery and trauma of the periodontal tissues, including bone. Several products, which augment tissue regeneration, have been manufactured and assayed in clinical practice with positive results. Emdogain is a recent addition in this field, as a tissue-regenerating product. The substance is a derivative of amelogenin, obtained from porcine embryonic tissues. At the present time, it is not known whether the substance can induce a local (due to the uptake of the substance) or systemic immune response. The aim of the present study was to evaluate, in vitro, the ability of Emdogain to influence, in vitro, the immune system. Peripheral blood lymphocytes, isolated for 10 healthy donors, were cultured in the presence of various concentrations of the substance, in order to determine the rate of cell proliferation, the expression of surface antigens and the production of cytokines and immunoglobulins. Under our experimental conditions, Emdogain produced a slight increase of the proliferation of lymphocytes, restricted to the CD25 (IL-2 receptor) fraction of the CD4 positive T-lymphocytes, and a concomitant decrease of CD19 positive B-lymphocytes. Other cell fractions (CD8 positive T-cells, B-cells and NK-cells) were not affected. Under our conditions too, immunoglobulin and cytokin (IL-2 and IL-6) production was not modified, even after a 3-day application of concentrations much higher than those used in clinical practice. Our data suggest that Emdogain slightly induce an immune response, restricted to the activated fraction of CD4 T-lymphocytes in vitro.

Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions.

Paxillin is a focal adhesion adapter protein involved in integrin signaling. Paxillin LD motifs bind several focal adhesion proteins including the focal adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-kinase linker, and the newly identified actin-binding protein actopaxin. Microsequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding protein revealed 100% identity with integrin-linked kinase (ILK)-1, a serine/threonine kinase that has been implicated in integrin, growth factor, and Wnt signaling pathways. Cloning of ILK from rat smooth muscle cells generated a cDNA that exhibited 99.6% identity at the amino acid level with human ILK-1. A monoclonal antibody raised against a region of the carboxyl terminus of ILK, which is identical in rat and human ILK-1 protein, recognized a 50-kDa protein in all cultured cells and tissues examined. Binding experiments showed that ILK binds directly to the paxillin LD1 motif in vitro. Co-immunoprecipitation from fibroblasts confirmed that the association between paxillin and ILK occurs in vivo in both adherent cells and cells in suspension. Immunofluorescence microscopy of fibroblasts demonstrated that endogenous ILK as well as transfected green fluorescent protein-ILK co-localizes with paxillin in focal adhesions. Analysis of the deduced amino acid sequence of ILK identified a paxillin-binding subdomain in the carboxyl terminus of ILK. In contrast to wild-type ILK, paxillin-binding subdomain mutants of ILK were unable to bind to the paxillin LD1 motif in vitro and failed to localize to focal adhesions. Thus, paxillin binding is necessary for efficient focal adhesion targeting of ILK and may therefore impact the role of ILK in integrin-mediated signal transduction events.

Experimental analysis of heart rate variability of long-recording electrocardiograms in normal subjects and patients with coronary artery disease and normal left ventricular function.

The heart rate signal contains valuable information about cardiac health, which cannot be extracted without the use of appropriate computerized methods. This paper presents an analysis of various electrocardiograms, the aim of which is to categorize them into two distinct groups. Group A represents young male subjects with no prior occurrence of coronary disease events and Group B represents middle-aged male subjects who have symptomatic coronary artery disease without myocardial infarction and whose 12-lead ECGs do not contain any abnormalities, thus wrongly indicating a normal subject. Electrocardiographic recordings are approximately 2h in length and acquired under conditions that favor the stationarity of collected data. Linear and nonlinear characteristics are studied by applying several techniques including Fourier analysis, Correlation Dimension Estimation, Approximate Entropy, and the Discrete Wavelet Transform. The small variations of the diagnostic information given by each one of the methods as well as the slightly different conclusions among similar studies indicate the necessity of further investigation, combined use, and complementary application of different approaches.

Safety of the ArF193 excimer laser for the removal of dental plaque and calculi: an in vitro histological study.

OBJECTIVE: To assay the safety of the ArF excimer laser in the integrity of human pulp elements. BACKGROUND DATA: The use of lasers in dentistry remains controversial, in spite of their increasing application in medical practice. The main reason for this discrepancy is the frequent report of damage to surrounding tissues and the dental pulp, due to the energy transfer, from the site of laser impact. The progress made on laser technology during the last 10 years, could overcome this obstacle and allow the use of lasers in dentistry. METHODS AND RESULTS: The present study reports the use of the ArF 193 excimer laser, under conditions of strict control of frequency and fluency, for the ablation of dental carries, plaque, and calculi, by the use of a new, articulated arm. We have tested 10 teeth, extracted for prosthetic reasons, immediately after extraction. Our in vitro results show that the ArF193 excimer laser does not produce any harm to the dental pulp (at least at the photo- or electronic microscopy level), whereas in a matter of seconds, it can be effective in removing all dental deposits. In addition, the use of the flexible articulated arm, makes this treatment comfortable and easier for both the dentist and patient. CONCLUSION: Under a strict control of laser technology, and the use of the new articulated arm presented, the use of the ArF excimer laser in dentistry is safe and comfortable.