Real Time Assays for Quantifying Cytotoxicity with Single Cell Resolution

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.     

A Microscope Automated Fluidic System to Study Bacterial Processes in Real Time

Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.     

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.     

The Effect of a Single Session of Short Duration Heart Rate Variability Biofeedback on EEG: A Pilot Study

This pilot study examines the effect of heart rate variability (HRV) biofeedback on measures of electroencephalogram (EEG) during and immediately after biofeedback. Eighteen healthy males exposed to work-related stress, were randomised into an HRV biofeedback (BIO) or a comparative group (COM). EEG was recorded during the intervention and during rest periods before and after the intervention. Power spectral density in theta, alpha and beta frequency bands and theta/beta ratios were calculated. During the intervention, the BIO group had higher relative theta power [Fz and Pz (p < 0.01), Cz (p < 0.05)], lower fronto–central relative beta power (p < 0.05), and higher theta/beta [Fz and Cz (p < 0.01), Pz (p < 0.05)] than the COM group. The groups showed different responses after the intervention with increased posterior theta/beta (p < 0.05) in the BIO group and altered posterior relative theta (p < 0.05), central relative beta (p = 0.06) and central–posterior theta/beta (p < 0.01) in the post-intervention rest period. The findings of this study suggest that a single session of HRV biofeedback after a single training session was associated with changes in EEG suggestive of increased internal attention and relaxation both during and after the intervention. However, the comparative intervention was associated with changes suggestive of increased mental effort and possible anxiety during and after the intervention.     

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.     

Attention and regulation of EEG alpha-attenuation responses

Two experiments with 16 normal adults of both sexes tested the hypothesis that inattention to a biofeedback display is associated with increased variability of those physiological processes that had been regulated by the biofeedback. Each experiment was a repeated-measures-on-independent-subjects-design. Dependent variables were the time durations and the mean rms power of two mutually exclusive segments of the parietal-occipital EEG: alpha and not-alpha segments. Independent variables were combination of counting tasks and instructions to look at, listen to, and count visual and auditory flashes and clicks. The durations of alpha and not-alpha segments were controlled or regulated by means of an alpha-contingent visual feedback stimulus, Attention to the feedback stimulus was challenged by instructions to count other, noncontingent stimuli. Control of alpha and not-alpha segments was least for conditions of (1) “sham” feedback, and (2) feedback with instructions to count noncontingent auditory clicks, which were presented 3/sec while the feedback visual stimuli were occurring. A new EEG test of attention and distraction was suggested.     

Real Time Observation of Single Membrane Protein Insertion Events by the Escherichia coli Insertase YidC

Membrane protein translocation and insertion is a central issue in biology. Here we focus on a minimal system, the membrane insertase YidC of Escherichia coli that inserts small proteins into the cytoplasmic membrane. In a reconstituted system individual insertion processes were followed by single-pair fluorescence resonance energy transfer (FRET), with a pair of fluorophores on YidC and the substrate Pf3 coat protein. After addition of N-terminally labeled Pf3 coat protein a close contact to YidC at its cytoplasmic label was observed. This allowed to monitor the translocation of the N-terminal domain of Pf3 coat protein across the membrane in real time. Translocation occurred within milliseconds as the label on the N-terminal domain rapidly approached the fluorophore on the periplasmic domain of YidC at the trans side of the membrane. After the close contact, the two fluorophores separated, reflecting the release of the translocated Pf3 coat protein from YidC into the membrane bilayer. When the Pf3 coat protein was labeled C-terminally, no translocation of the label was observed although efficient binding to the cytoplasmic positions of YidC occurred.     

From Single Cells to Tissues: Interactions between the Matrix and Human Breast Cells in Real Time

Background

Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.

Results

The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.

Conclusions

Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.     

Attention modulation regulates both motor and non-motor performance: a high-density EEG study in Parkinson's disease

We have previously shown that, in early stages of Parkinson's disease (PD), patients with higher reaction times are also more impaired in visual sequence learning, suggesting that movement preparation shares resources with the learning of visuospatial sequences. Here, we ascertained whether, in patients with PD, the pattern of the neural correlates of attentional processes of movement planning predict sequence learning and working memory abilities. High density Electroencephalography (EEG, 256 electrodes) was recorded in 19 patients with PD performing reaching movements in a choice reaction time paradigm. Patients were also tested with Digit Span and performed a visuomotor sequence learning task that has an important declarative learning component. We found that attenuation of alpha/beta oscillatory activity before the stimulus presentation in frontoparietal regions significantly correlated with reaction time in the choice reaction time task, similarly to what we had previously found in normal subjects. In addition, such activity significantly predicted the declarative indices of sequence learning and the scores in the Digit Span task. These findings suggest that some motor and non motor PD signs might have common neural bases, and thus, might have a similar response to the same behavioral therapy. In addition, these results might help in designing and testing the efficacy of novel rehabilitative approaches to improve specific aspects of motor performance in PD and other neurological disorders.     

Schizophrenia Detection and Classification by Advanced Analysis of EEG Recordings Using a Single Electrode Approach

Electroencephalographic (EEG) analysis has emerged as a powerful tool for brain state interpretation and diagnosis, but not for the diagnosis of mental disorders; this may be explained by its low spatial resolution or depth sensitivity. This paper concerns the diagnosis of schizophrenia using EEG, which currently suffers from several cardinal problems: it heavily depends on assumptions, conditions and prior knowledge regarding the patient. Additionally, the diagnostic experiments take hours, and the accuracy of the analysis is low or unreliable. This article presents the “TFFO” (Time-Frequency transformation followed by Feature-Optimization), a novel approach for schizophrenia detection showing great success in classification accuracy with no false positives. The methodology is designed for single electrode recording, and it attempts to make the data acquisition process feasible and quick for most patients.     

Analysis of the Emotional-Cognitive Processes using a Modified Multisource Interference Task and Recording of EEG and Behavioral Responses

Neurophysiology - We investigated changes in the EEG signals and the latency of behavioral responses in the cognitive test with interfering emotional stimuli. It was hypothesized that the cognitive...     

Effect of EEG Biofeedback on Cognitive Flexibility in Children with Attention Deficit Hyperactivity Disorder With and Without Epilepsy

Attention deficit hyperactivity disorder (ADHD) is one of the most common developmental disorders in school-aged children. Symptoms consistent with ADHD have been observed in 8–77 % of children with epilepsy. Researchers have been motivated to search for alternative forms of treatment because 30 % of patients with ADHD cannot be treated by psychostimulants. Several studies support the use of a multimodal treatment approach that includes neurofeedback (NF) for the long-term management of ADHD. These studies have shown that NF provides a sustained effect, even without concurrent treatment with stimulants. We aimed to assess cognitive flexibility in ADHD children with and without temporal lobe epilepsy (TLE), and to evaluate the effects of NF on cognitive flexibility in these groups of children. We prospectively evaluated 69 patients with ADHD aged 9–12 years. The control group was 26 ADHD children without TLE who received no treatment. The first experimental group comprised 18 children with ADHD. The second experimental group comprised 25 age-matched ADHD children with TLE. This group was further divided in two subgroups. One subgroup comprised those with mesial temporal lobe epilepsy (16 patients, 9 with hippocampal sclerosis and 7 with hippocampal atrophy), and the other with lateral temporal lobe epilepsy (9 patients, 5 with temporal lobe dysplasia, 3 with temporal lobe cysts, and 1 with a temporal lobe cavernoma). We treated their ADHD by conducting 30 sessions of EEG NF. Reaction time and error rates on the Trail Making Test Part B were compared before and after treatment, and significant differences were found for all groups of patients except those who had mesial temporal lobe epilepsy with hippocampal atrophy. Our results demonstrate that in most cases, NF can be considered an alternative treatment option for ADHD children even if they have TLE. Additional studies are needed to confirm our results.     

Attention and eye movements in human: psychophysiological concepts, neurophysiological models and EEG correlates

A review. Recently published articles concerning the problem of attention are discussed, the most popular psychophysiological concepts and neurophysiological models of attention are described, and correlation of spatial attention and saccadic eyes movements is shown. The evidence for reflection of attention mechanisms and saccade preparation in intensity and topography of the visual evoked potentials and event-related potentials is given. On the basis of the results obtained by the authors and literature data, the contribution of attention to preparation of a saccade and its programming is shown. Different kinds of attention are reflected in a complex of EEG potentials of various duration and polarity. The analysis of parameters and topography of these potentials can serve a tool for investigation of the attention mechanisms.     

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation

In the present work, we demonstrate a method for concurrent collection of EEG/fMRI data. In our setup, EEG data are collected using a high-density 256-channel sensor net. The EEG amplifier itself is contained in a field isolation containment system (FICS), and MRI clock signals are synchronized with EEG data collection for subsequent MR artifact characterization and removal. We demonstrate this method first for resting state data collection. Thereafter, we demonstrate a protocol for EEG/fMRI data recording, while subjects listen to a tape asking them to visualize that their left hand is immersed in a cold-water bath and referred to, here, as the cold glove paradigm. Thermal differentials between each hand are measured throughout EEG/fMRI data collection using an MR compatible temperature sensor that we developed for this purpose. We collect cold glove EEG/fMRI data along with simultaneous differential hand temperature measurements both before and after hypnotic induction. Between pre and post sessions, single modality EEG data are collected during the hypnotic induction and depth assessment process. Our representative results demonstrate that significant changes in the EEG power spectrum can be measured during hypnotic induction, and that hand temperature changes during the cold glove paradigm can be detected rapidly using our MR compatible differential thermometry device.     

Changes in the Central Regulation of the Respiratory Function after a Single Session of Intermittent Normobaric Hypoxia

Correlations between EEG findings and external respiration and gas exchange indices recorded prior to and after a single session of intermittent normobaric hypoxia were calculated by means of the Neirokartograf (MBN) program. Changes in the central regulation of respiration were observed for a period of more than one hour. The hypoxia caused a decline in EEG coherence in the left hemisphere and a decrease in the statistically significant correlations between the bioelectric rhythms of the , , and ranges and the external respiration and gas exchange indices.     

The Effects of a Single Session of Upper Alpha Neurofeedback for Cognitive Enhancement: A Sham-Controlled Study

The minimization of the non-specific factors of neurofeedback (NF) is an important aspect to further advance in the understanding of the effects of these types of procedures. This paper investigates the NF effects of a single session (25 min) of individual upper alpha enhancement following a sham-controlled experimental design (19 healthy participants). We measured immediate effects after the training and 1-day lasting EEG effects (eyes closed resting state and task-related activity), as well as the event-locked EEG effects during the execution of a mental rotation task. These metrics were computed in trained (upper alpha) and non-trained EEG parameters (lower alpha and lower beta). Several cognitive functions were assessed such as working memory and mental rotation abilities. The NF group showed increased upper alpha power after training in task-related activity (not significantly sustained 1 day after) and higher pre-stimulus power during the mental rotation task. Both groups improved cognitive performance, with a more prominent improvement for the NF group, however a single session seems to be insufficient to yield significant differences between groups. A higher number of training sessions seems necessary to achieve long-lasting effects on the electrophysiology and to enhance the behavioral effects.     

EEG Coherence as an Indicator of Integrative Brain Processes in Internet and Game Addiction

Human Physiology - A coherence analysis of Internet and game addicts was carried out. The results of studies indicate changes in the functional interaction of various cortical zones in both...