Estimation of phase in EEG rhythms for real-time applications.

OBJECTIVE: Estimating the ongoing phase of oscillations in electroencephalography (EEG) recordings is an important aspect of understanding brain function, as well as for the development of phase-dependent closed-loop real-time systems that deliver stimuli. Such stimuli may take the form of direct brain stimulation (for example transcranial magnetic stimulation), or sensory stimuli (for example presentation of an auditory stimulus). We identify two linked problems related to estimating the phase of EEG rhythms with a specific focus on the alpha-band: 1) when the signal after a specific stimulus is unknown (real-time case), or 2) when it is corrupted by the presence of the stimulus itself (offline analysis). We propose methods to estimate the phase at the presentation time of these stimuli. APPROACH: Machine learning methods are used to learn the causal mapping from an unprocessed EEG recording to a phase estimate generated with a non-causal signal processing chain. This mapping is then used to predict the phase causally where non-causal methods are inappropriate. MAIN RESULTS: We demonstrate the ability of these machine learning methods to estimate instantaneous phase from an EEG signal subjected to very minor pre-processing with higher accuracy than commonly used signal-processing methods. SIGNIFICANCE: Neural oscillations have been implicated in a wide variety of sensory, cognitive and motor functions. The instantaneous phase of these rhythms may reflect specific processes of computation which can be acted upon if they can be estimated with sufficient accuracy. Such brain-state dependent paradigms are of increasing medical and scientific interest.

Do E-health interventions improve physical activity in young people: a systematic review.

OBJECTIVES: This study aims to review the current literature to assess the effectiveness of E-health interventions in increasing physical activity (PA) in young people. STUDY DESIGN: This study is a systematic review of the literature. METHODS: A search of the literature databases Embase, MEDLINE and the Cochrane Library using key words 'Adolescents'; 'Young people'; 'Students'; 'Young Adults'; 'Teenagers'; 'E-health'; 'Internet-based'; 'Web-based'; 'Exercise'; 'Activity'; 'Sport' and 'Intervention' yielded 10 articles which fit the criteria for inclusion. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol was used, and papers were excluded if they were disease focused, not specific to young people (those attending school, college or university) or did not measure PA as an outcome. RESULTS: Eight of the 10 studies had significant increases in PA as a result of an E-health intervention. Studies that did not use a theoretical principle to underpin their intervention did not achieve successful results. Interventions based on social cognitive theory were very successful in achieving an increase in PA. The theory of planned behaviour had mixed results, with studies having contrasting results. Specific, measurable, achievable, relevant and time-bound (SMART) goal principle was not effective in increasing PA but had positive findings in supplementary outcomes such as goal setting. CONCLUSIONS: E-health interventions are a very successful way to increase PA. More research is required to look at what theoretical principles are best to underpin interventions and also to assess the length of intervention required for optimal results after intervention. Ideas surrounding implementation and the mediums used require more studies to evidence base these interventions for schools, colleges and university via intracurriculum or extracurriculum.

Metallothionein as a clonable tag for protein localization by electron microscopy of cells.

A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.

Effects of caffeine on session ratings of perceived exertion.

This study examined effects of caffeine on session ratings of perceived exertion (RPE) following 30 min constant-load cycling. Individuals (n = 15) of varying aerobic fitness completed a [Formula: see text] max trial and two 30 min cycling bouts (double-blind, counterbalanced) following ingestion of 6 mL/kg of caffeine or matched placebo. RPE overall, legs and breathing were estimated every 5 min and session RPE was estimated 30 min post-exercise using the OMNI pictorial scale. Session RPE for caffeine and placebo trails were compared using paired t test. Between-trial comparisons of HR, RPE overall, RPE legs and RPE breathing were analyzed using an independent 2 (trial) × 6 (time point) repeated measures analysis of variance (ANOVA) for each dependent variable. Caffeine resulted in a significantly lower session RPE (p < 0.05) for caffeine (6.1 ± 2.2) versus placebo (6.8 ± 2.1). Acute perceptual responses were significantly lower for caffeine for RPE overall (15, 20, 25, and 30 min), RPE breathing (15, 20, 25, and 30 min) and RPE legs (20 and 30 min). Survey responses post-exercise revealed greater feelings of nervousness, tremors, restlessness and stomach distress following caffeine versus placebo. Blunted acute RPE and survey responses suggest participants responded to caffeine ingestion. Caffeine decreased acute RPE during exercise which could partially account for lower session RPE responses. However, decreased session RPE could also reveal a latent analgesic affect of caffeine extending into recovery. Extending the understanding of session RPE could benefit coaches in avoiding overtraining when adjusting training programs.

Fabrication and characterization of a carbon nanotube-based nanoknife.

We demonstrate the fabrication and testing of a prototype microtome knife based on a multiwalled carbon nanotube (MWCNT) for cutting approximately 100 nm thick slices of frozen-hydrated biological samples. A piezoelectric-based 3D manipulator was used inside a scanning electron microscope (SEM) to select and position individual MWCNTs, which were subsequently welded in place using electron beam-induced deposition. The knife is built on a pair of tungsten needles with provision to adjust the distance between the needle tips, accommodating various lengths of MWCNTs. We performed experiments to test the mechanical strength of a MWCNT in the completed device using an atomic force microscope tip. An increasing force was applied at the mid-point of the nanotube until failure occurred, which was observed in situ in the SEM. The maximum breaking force was approximately (8 x 10(-7)) N which corresponds well with the typical microtome cutting forces reported in the literature. In situ cutting experiments were performed on a cell biological embedding plastic (epoxy) by pushing it against the nanotube. Initial experiments show indentation marks on the epoxy surface. Quantitative analysis is currently limited by the surface asperities, which have the same dimensions as the nanotube.

Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms.

Mitotic chromosomes segregate at the ends of shortening spindle microtubules (MTs). In budding yeast, the Dam1 multiprotein complex supports this dynamic attachment, thereby contributing to accurate chromosome segregation. Purified Dam1 will track the end of a depolymerizing MT and can couple it to microbead transport in vitro. The processivity of such motions has been thought to depend on rings that the Dam1 complex can form around MTs, but the possibility that alternative coupling geometries contribute to these motilities has not been considered. Here, we demonstrate that both rings and nonencircling Dam1 oligomers can track MT ends and enable processive cargo movement in vitro. The coupling properties of these two assemblies are, however, quite different, so each may make a distinct contribution to chromosome motility.

Force production by depolymerizing microtubules: a theoretical study.

Chromosome movement during mitosis is powered in part by energy released through the depolymerization of kinetochore microtubules (MTs). Strong but indirect evidence suggests the existence of a specialized coupling between kinetochores and MT plus ends that enables this transduction of chemical energy into mechanical work. Analysis of this phenomenon is important for learning how energy is stored within the MT lattice, how it is transduced, and how efficient the process can be, given coupling devices of different designs. Here we use a recently developed molecular-mechanical model of MTs to examine the mechanism of disassembly dependent force generation. Our approach is based on changes in tubulin dimer conformation that occur during MT disassembly. We find that all of the energy of polymerization-associated GTP hydrolysis can be stored as deformations of the longitudinal bonds between tubulin dimers, and its optimal use does not require the weakening of lateral bonds between dimers. Maximum utilization of this stored energy and, hence, the generation of the strongest possible force, is achieved by a protofilament power-stroke mechanism, so long as the coupling device does not restrict full dissociation of the lateral bonds between tubulin dimers.

Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival.

Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion. The essential function is believed to result from its role as a Pol1p (polymerase alpha) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p. In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage. Characterization of an inducible allele of mcl1+, (nmt41)mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Delta, rqh1Delta, and hsk1-1312. Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea. We take these data to mean that Mcl1p is a dynamic component of the polymerase alpha complex during replication and is important for the replication stress response in fission yeast.

The use of filter membranes for high-pressure freezing of cell monolayers.

Rapid freezing of cells and tissues, followed by freeze-substitution fixation and plastic embedding, has become a highly reliable method for preparing samples for imaging in the electron microscope. High-pressure freezing is an efficient means of immobilizing suspensions of yeasts, thick pellets of mammalian cells, or small (< 0.5 mm) pieces of plant or animal tissue. Monolayers of cultured mammalian cells that are too thick for efficient immobilization by other modes of rapid freezing have also been successfully preserved by this method. Monolayer cultures are often important because they can be imaged by light microscopy (LM) both before and after their preparation for electron microscopy (EM). Additionally, some monolayer cultures serve as model systems for physiological processes, so it is important that cells under study can grow on a substrate that is both physiologically appropriate and convenient for EM processing. Here we describe a reliable method for preparing mammalian cell monolayers (PtK1 and polarized MDCK) for EM. Our protocol results in good preservation of cellular ultrastructure, it is a useful companion to studies of cell physioloy and, with some limitation, is suitable for correlative LM and EM.

Two related kinesins, klp5+ and klp6+, foster microtubule disassembly and are required for meiosis in fission yeast.

The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity. We have characterized two related kinesins in fission yeast, klp5+ and klp6+,, that are amino-terminal motors of the KIP3 subfamily. Analysis of null mutants demonstrates that neither klp5+ nor klp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior. klp5Delta and klp6Delta are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell. This phenotype is greatly enhanced in the cell cycle mutant cdc25-22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest. Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent. During the meiotic phase of the life cycle, both of these kinesins are essential. Spore viability is low in homozygous crosses of either null mutant. Heterozygous crosses of klp5Delta with klp6Delta have an intermediate viability, suggesting cooperation between these proteins in meiosis.

pkl1(+)and klp2(+): Two kinesins of the Kar3 subfamily in fission yeast perform different functions in both mitosis and meiosis.

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.

The domain structure of centromeres is conserved from fission yeast to humans.

The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.

Structural evidence for multiple transport mechanisms through the Golgi in the pancreatic beta-cell line, HIT-T15.

Accurate data on the three-dimensional architecture of the Golgi is prerequisite for evaluating the mechanisms of transit through this organelle. Here we detail the structure of the Golgi ribbon within part of an insulin-secreting cell in three dimensions at approximately 6 nm resolution. Rapid freezing, freeze-substitution and electron tomography were employed. The Golgi in this region is composed of seven cisternae. The cis-most element is structurally intermediate between the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-most cisterna characterized in three dimensions at high resolution in a normal rat kidney cell [Ladinsky, Mastronarde, McIntosh, Howell and Staehelin (1999) J. Cell Biol. 144, 1135-1149]. There are three trans-cisternae that demonstrate morphological and functional variation. The membrane surface areas and volumes of these elements decrease from cis to trans. The two trans-most cisternae are dissociated from the stack and are fragmented by tubulation. ER closely adheres to and inserts between individual trans-cisternae. Many of the 2119 small, clathrin-negative vesicles that are in close proximity to the Golgi fill the region where trans-cisternae have moved out of register with the ribbon. These data provide evidence that cisternal progression/maturation, trafficking via membrane tubules and vesicle-mediated transport act in concert in the same region of the Golgi ribbon, and suggest an important role for the ER in regulating membrane dynamics at the trans-Golgi.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography.

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at approximately 6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 x 3.2 x 1.2 microm(3)) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455-476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, approximately 66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Tea2p is a kinesin-like protein required to generate polarized growth in fission yeast.

Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2(+), that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Delta cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.

CENP-meta, an essential kinetochore kinesin required for the maintenance of metaphase chromosome alignment in Drosophila.

CENP-meta has been identified as an essential, kinesin-like motor protein in Drosophila. The 257-kD CENP-meta protein is most similar to the vertebrate kinetochore-associated kinesin-like protein CENP-E, and like CENP-E, is shown to be a component of centromeric/kinetochore regions of Drosophila chromosomes. However, unlike CENP-E, which leaves the centromere/kinetochore region at the end of anaphase A, the CENP-meta protein remains associated with the centromeric/kinetochore region of the chromosome during all stages of the Drosophila cell cycle. P-element-mediated disruption of the CENP-meta gene leads to late larval/pupal stage lethality with incomplete chromosome alignment at metaphase. Complete removal of CENP-meta from the female germline leads to lethality in early embryos resulting from defects in metaphase chromosome alignment. Real-time imaging of these mutants with GFP-labeled chromosomes demonstrates that CENP-meta is required for the maintenance of chromosomes at the metaphase plate, demonstrating that the functions required to establish and maintain chromosome congression have distinguishable requirements.

Life cycles of yeast spindle pole bodies: getting microtubules into a closed nucleus.

The spindle pole body (SPB) is the principal microtubule organizing center of budding and fission yeast. We have examined SPBs and their associated microtubules from both organisms, using electron microscopy and three-dimensional reconstruction techniques, to identify the structural changes that accompany progression through the cell cycle. In this report, we compare these changes in the two kinds of yeasts and present a model for how microtubules get into a closed nucleus.