Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of PSEP are not well understood, especially the intermediate processes leading to delivery. PSEP is an electrical method, yet the relationship between PSEP and electrical impedance remains underexplored. In this study, a device capable of measuring impedance and performing PSEP is developed and the changes in transepithelial electrical impedance (TEEI) are monitored. These measurements show TEEI increases following PSEP, unlike other electroporation methods. The authors then demonstrate how cell culture conditions and electrical waveforms influence this response. More importantly, TEEI response features are correlated with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which the authors expect will aid PSEP optimization for new cell types and cargos.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of the PSEP delivery process are not well understood, partly because most PSEP studies rely solely on imaging for evaluating delivery. Although effective, imaging alone limits understanding of intermediate processes leading to delivery. PSEP is an electrical process, so electrical impedance measurements naturally complement imaging for PSEP characterization. In this study, we developed a device capable of measuring impedance and performing PSEP and we monitored changes in transepithelial electrical impedance (TEEI). Our measurements show TEEI increases following PSEP, unlike other electroporation methods. We then demonstrated how cell culture conditions and electrical waveforms influence this response. More importantly, we correlated TEEI response features with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which we expect will aid PSEP optimization for new cell types and cargos.

Neural and behavioral adaptations to frontal theta neurofeedback training: A proof of concept study.

Previous neurofeedback research has shown training-related frontal theta increases and performance improvements on some executive tasks in real feedback versus sham control groups. However, typical sham control groups receive false or non-contingent feedback, making it difficult to know whether observed differences between groups are associated with accurate contingent feedback or other cognitive mechanisms (motivation, control strategies, attentional engagement, fatigue, etc.). To address this question, we investigated differences between two frontal theta training groups, each receiving accurate contingent feedback, but with different top-down goals: (1) increase and (2) alternate increase/decrease. We hypothesized that the increase group would exhibit greater increases in frontal theta compared to the alternate group, which would exhibit lower frontal theta during down- versus up-modulation blocks over sessions. We also hypothesized that the alternate group would exhibit greater performance improvements on a Go-NoGo shooting task requiring alterations in behavioral activation and inhibition, as the alternate group would be trained with greater task specificity, suggesting that receiving accurate contingent feedback may be the more salient learning mechanism underlying frontal theta neurofeedback training gains. Thirty young healthy volunteers were randomly assigned to increase or alternate groups. Training consisted of an orientation session, five neurofeedback training sessions (six blocks of six 30-s trials of FCz theta modulation (4-7 Hz) separated by 10-s rest intervals), and six Go-NoGo testing sessions (four blocks of 90 trials in both Low and High time-stress conditions). Multilevel modeling revealed greater frontal theta increases in the alternate group over training sessions. Further, Go-NoGo task performance increased at a greater rate in the increase group (accuracy and reaction time, but not commission errors). Overall, these results reject our hypotheses and suggest that changes in frontal theta and performance outcomes were not explained by reinforcement learning afforded by accurate contingent feedback. We discuss our findings in terms of alternative conceptual and methodological considerations, as well as limitations of this research.

Two-Photon Polymerized Shape Memory Microfibers: A New Mechanical Characterization Method in Liquid.

Two-photon polymerization (TPP) has been widely used to create 3D micro- and nanoscale scaffolds for biological and mechanobiological studies, which often require the mechanical characterization of the TPP fabricated structures. To satisfy physiological requirements, most of the mechanical characterizations need to be conducted in liquid. However, previous characterizations of TPP fabricated structures were all conducted in air due to the limitation of conventional micro- and nanoscale mechanical testing methods. In this study, we report a new experimental method for testing the mechanical properties of TPP-printed microfibers in liquid. The experiments show that the mechanical behaviors of the microfibers tested in liquid are significantly different from those tested in air. By controlling the TPP writing parameters, the mechanical properties of the microfibers can be tailored over a wide range to meet a variety of mechanobiology applications. In addition, it is found that, in water, the plasticly deformed microfibers can return to their pre-deformed shape after tensile strain is released. The shape recovery time is dependent on the size of microfibers. The experimental method represents a significant advancement in mechanical testing of TPP fabricated structures and may help release the full potential of TPP fabricated 3D tissue scaffold for mechanobiological studies.

Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality.

Complex patterns of hemodynamic wall shear stress occur in regions of arterial branching and curvature. Areas within these regions can be highly susceptible to atherosclerosis. Although many studies have characterized the response of vascular endothelial cells to shear stress in a categorical manner, our study herein addresses the need of characterizing endothelial behaviors over a continuous range of shear stress conditions that reflect the extensive variations seen in the vasculature. We evaluated the response of human umbilical vein endothelial cell monolayers to orbital flow at 120, 250, and 350 revolutions per minute (RPM) for 24 and 72 h. The orbital shaker model uniquely provides a continuous range of shear stress conditions from low and multidirectional at the center of each well of a culture plate to high and unidirectional at the periphery. We found distinct patterns of endothelial nuclear area, nuclear major and minor diameters, nuclear aspect ratio, and expression of endothelial nitric oxide synthase over this range of shear conditions and relationships were fit with linear and, where appropriate, power functions. Nuclear area was particularly sensitive with increases in the low and multidirectional WSS region that incrementally decreased as WSS became higher in magnitude and more unidirectional over the radius of the cell layers. The patterns of all endothelial behaviors exhibited high correlations (positive and negative) with metrics of shear stress magnitude and directionality that have been shown to strongly associate with atherosclerosis. Our findings demonstrate the exquisite sensitivity of these endothelial behaviors to incremental changes in shear stress magnitude and directionality, and provide critical quantitation of these relationships for predicting the susceptibility of an arterial segment to diseases such as atherosclerosis, particularly within complex flow environments in the vasculature such as around bifurcations.

A pilot study to understand the relationship between cortical arousals and leg movements during sleep.

Leg movements during sleep occur in patients with sleep pathology and healthy individuals. Some (but not all) leg movements during sleep are related to cortical arousals which occur without conscious awareness but have a significant effect of sleep fragmentation. Detecting leg movements during sleep that are associated with cortical arousals can provide unique insight into the nature and quality of sleep. In this study, a novel leg movement monitor that uses a unique capacitive displacement sensor and 6-axis inertial measurement unit, is used in conjunction with polysomnography to understand the relationship between leg movement and electroencephalogram (EEG) defined cortical arousals. In an approach that we call neuro-extremity analysis, directed connectivity metrics are used to interrogate causal linkages between EEG and leg movements measured by the leg movement sensors. The capacitive displacement measures were more closely related to EEG-defined cortical arousals than inertial measurements. Second, the neuro-extremity analysis reveals a temporally evolving connectivity pattern that is consistent with a model of cortical arousals in which brainstem dysfunction leads to near-instantaneous leg movements and a delayed, filtered signal to the cortex leading to the cortical arousal during sleep.

An equivalent circuit model for localized electroporation on porous substrates.

In vitro intracellular delivery is a fundamental challenge with no widely adopted methods capable of both delivering to millions of cells and controlling that delivery to a high degree of accuracy. One promising method is porous substrate electroporation (PSEP), where cells are cultured on porous substrates and electric fields are used to permeabilize discrete portions of the cell membrane for delivery. A major obstacle to the widespread use of PSEP is a poor understanding of the various impedances that constitute the system, including the impedances of the porous substrate and the cell monolayer, and how these impedances are influenced by experimental parameters. In response, we used impedance measurements to develop an equivalent circuit model that closely mimics the behavior of each of the main components of the PSEP system. This circuit model reveals for the first time the distribution of voltage across the electrode-electrolyte interface impedances, the channels of the porous substrate, the cell monolayer, and the transmembrane potential during PSEP. We applied sample waveforms through our model to understand how waveforms can be improved for future studies. Our model was validated from intracellular delivery of protein using PSEP.

Tasimelteon safely and effectively improves sleep in Smith-Magenis syndrome: a double-blind randomized trial followed by an open-label extension.

PURPOSE: To assess the efficacy of tasimelteon to improve sleep in Smith-Magenis syndrome (SMS). METHODS: A 9-week, double-blind, randomized, two-period crossover study was conducted at four US clinical centers. Genetically confirmed patients with SMS, aged 3 to 39, with sleep complaints participated in the study. Patients were assigned to treatment with tasimelteon or placebo in a 4-week crossover study with a 1-week washout between treatments. Eligible patients participated in an open-label study and were followed for >3 months. RESULTS: Improvement of sleep quality (DDSQ50) and total sleep time (DDTST50) on the worst 50% of nights were primary endpoints. Secondary measures included actigraphy and behavioral parameters. Over three years, 52 patients were screened, and 25 patients completed the randomized portion of the study. DDSQ50 significantly improved over placebo (0.4, p = 0.0139), and DDTST50 also improved (18.5 minutes, p = 0.0556). Average sleep quality (0.3, p = 0.0155) and actigraphy-based total sleep time (21.1 minutes, p = 0.0134) improved significantly, consistent with the primary outcomes. Patients treated for ≥90 days in the open-label study showed persistent efficacy. Adverse events were similar between placebo and tasimelteon. CONCLUSION: Tasimelteon safely and effectively improved sleep in SMS.

A physiological and dynamical systems model of stress.

Stress responses vary drastically for a given set of stimuli, individuals, or points in time. A potential source of this variance that is not well characterized arises from the theory of stress as a dynamical system, which implies a complex, nonlinear relationship between environmental/situational inputs and the development/experience of stress. In this framework, stress vs. non-stress states exist as attractor basins in a physiologic phase space. Here, we develop a model of stress as a dynamical system by coupling closed loop physiologic control to a dynamic oscillator in an attractor landscape. By characterizing the evolution of this model through phase space, we demonstrate strong sensitivity to the parameters controlling the dynamics and demonstrate multiple features of stress responses found in current research, implying that these parameters may contribute to a significant source of variability observed in empiric stress research.

Interplay between state anxiety, heart rate variability, and cognition: An ex-Gaussian analysis of response times.

The present study employed an ex-Gaussian model of response times (RTs) to elucidate the cognitive processes related to experimentally induced state anxiety (SA) and vagally mediated heart rate variability (vmHRV), an indicator of adaptive responses in both cognitive and affective domains. Participants (n = 110) completed a dual task composed of (i) a flanker attention and (2) working memory load task, while SA was induced by threat of noise. Electrocardiography was measured during the dual task and during four baseline periods in order to calculate vmHRV. RTs on the flanker task were fit to an ex-Gaussian distribution, which estimated three RT parameters: mu (Gaussian mean), sigma (Gaussian SD), and tau (combination of exponential mean and SD). First, findings indicate that threat of noise was associated with reductions in mu and tau, suggesting that SA might improve attention and motor responding. Second, higher resting vmHRV was associated with lower tau (averaged across conditions) and stronger threat-related decreases in tau. Third, intra-individual decreases in vmHRV were accompanied by concomitant decreases in tau. These findings support roles for trait and state vagal control in guiding adaptive anxiety-related (and anxiety-unrelated) attentional responses. Findings are consistent with extant theories that emphasize functional interrelations among emotion, cognition, and vagal function.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery.

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general.

Microfluidic Device for Localized Electroporation.

Electroporation is a common method of transfection due to its relatively low risk and high transfection efficiency. The most common method of electroporation is bulk electroporation which is easily performed on large quantities of cells yet results in variable levels of viability and transfection efficiency across the population. Localized electroporation is an alternative that can be administered on a similar scale but results in much more consistent with higher quality transfection and higher cell viability. This chapter discusses the creation and use of a simple and cost-effective device using porous membrane for performing localized electroporation.

Linking Emotional Reactivity Between Laboratory Tasks and Immersive Environments Using Behavior and Physiology.

An event or experience can induce different emotional responses between individuals, including strong variability based on task parameters or environmental context. Physiological correlates of emotional reactivity, as well as related constructs of stress and anxiety, have been found across many physiological metrics, including heart rate and brain activity. However, the interdependances and interactions across contexts and between physiological systems are not well understood. Here, we recruited military and law enforcement to complete two experimental sessions across two different days. In the laboratory session, participants viewed high-arousal negative images while brain activity electroencephalogram (EEG) was recorded from the scalp, and functional connectivity was computed during the task and used as a predictor of emotional response during the other experimental session. In an immersive simulation session, participants performed a shoot-don't-shoot scenario while heart rate electrocardiography (ECG) was recorded. Our analysis examined the relationship between the sessions, including behavioral responses (emotional intensity ratings, task performance, and self-report anxiety) and physiology from different modalities [brain connectivity and heart rate variability (HRV)]. Results replicated previous research and found that behavioral performance was modulated within-session based on varying levels of emotional intensity in the laboratory session (t (24) = 4.062, p < 0.0005) and stress level in the simulation session (Z = 2.45, corrected p-value = 0.0142). Both behavior and physiology demonstrated cross-session relationships. Behaviorally, higher intensity ratings in the laboratory was related to higher self-report anxiety in the immersive simulation during low-stress (r = 0.465, N = 25, p = 0.019) and high-stress (r = 0.400, N = 25, p = 0.047) conditions. Physiologically, brain connectivity in the theta band during the laboratory session significantly predicted low-frequency HRV in the simulation session (p < 0.05); furthermore, a frontoparietal connection accounted for emotional intensity ratings during the attend laboratory condition (r = 0.486, p = 0.011) and self-report anxiety after the high-stress simulation condition (r = 0.389, p = 0.035). Interestingly, the predictive power of the brain activity occurred only for the conditions where participants had higher levels of emotional reactivity, stress, or anxiety. Taken together, our findings describe an integrated behavioral and physiological characterization of emotional reactivity.

Intra-Individual Variability in Vagal Control Is Associated With Response Inhibition Under Stress.

Dynamic intra-individual variability (IIV) in cardiac vagal control across multiple situations is believed to contribute to adaptive cognition under stress; however, a dearth of research has empirically tested this notion. To this end, we examined 25 U.S. Army Soldiers (all male, mean age = 30.73, standard deviation (SD) = 7.71) whose high-frequency heart rate variability (HF-HRV) was measured during a resting baseline and during three conditions of a shooting task (training, low stress, high stress). Response inhibition was measured as the correct rejection (CR) of friendly targets during the low and high stress conditions. We tested the association between the SD of HF-HRV across all four task conditions (IIV in vagal control) and changes in response inhibition between low and high stress. Greater differences in vagal control between conditions (larger IIV) were associated with higher tonic vagal control during rest, and stronger stress-related decreases in response inhibition. These results suggest that flexibility in vagal control is supported by tonic vagal control, but this flexibility also uniquely relates to adaptive cognition under stress. Findings are consistent with neurobehavioral and dynamical systems theories of vagal function.

Improving Real-Life Estimates of Emotion Based on Heart Rate: A Perspective on Taking Metabolic Heart Rate Into Account.

Extracting information about emotion from heart rate in real life is challenged by the concurrent effect of physical activity on heart rate caused by metabolic need. "Non-metabolic heart rate," which refers to the heart rate that is caused by factors other than physical activity, may be a more sensitive and more universally applicable correlate of emotion than heart rate itself. The aim of the present article is to explore the evidence that non-metabolic heart rate, as it has been determined up until now, indeed reflects emotion. We focus on methods using accelerometry since these sensors are readily available in devices suitable for daily life usage. The evidence that non-metabolic heart rate as determined by existing methods reflect emotion is limited. Alternative possible routes are explored. We conclude that for real-life cases, estimating the type and intensity of activities based on accelerometry (and other information), and in turn use those to determine the non-metabolic heart rate for emotion is most promising.

Different profiles of decision making and physiology under varying levels of stress in trained military personnel.

Decision making is one of the most vital processes we use every day, ranging from mundane decisions about what to eat to life-threatening choices such as how to avoid a car collision. Thus, the context in which our decisions are made is critical, and our physiology enables adaptive responses that account for how environmental stress influences our performance. The relationship between stress and decision making can additionally be affected by one's expertise in making decisions in high-threat environments, where experts can develop an adaptive response that mitigates the negative impacts of stress. In the present study, 26 male military personnel made friend/foe discriminations in an environment where we manipulated the level of stress. In the high-stress condition, participants received a shock when they incorrectly shot a friend or missed shooting a foe; in the low-stress condition, participants received a vibration for an incorrect decision. We characterized performance using signal detection theory to investigate whether a participant changed their decision criterion to avoid making an error. Results showed that under high-stress, participants made more false alarms, mistaking friends as foes, and this co-occurred with increased high frequency heart rate variability. Finally, we examined the relationship between decision making and physiology, and found that participants exhibited adaptive behavioral and physiological profiles under different stress levels. We interpret this adaptive profile as a marker of an expert's ingrained training that does not require top down control, suggesting a way that expert training in high-stress environments helps to buffer negative impacts of stress on performance.

Overlapping brain network and alpha power changes suggest visuospatial attention effects on driving performance.

When humans perform prolonged, continuous tasks, their performance fluctuates. The etiology of these fluctuations is multifactorial, but they are influenced by changes in attention reflected in underlying neural dynamics. Previous work with electroencephalography has suggested that prestimulus alpha power is a neural signature of attention allocation with higher power portending relatively poorer performance. The functional mechanisms subserving these changes in alpha power and behavior are postulated to be the result of networked neural activity that permits flexibility in the allocation of attention. Here, we directly examine the similarity between prestimulus alpha connectivity and power in relation to performance fluctuations in a continuous driving task. Participants were asked to maintain their vehicle in the center of a simulated highway, and we evaluated their performance by randomly perturbing the vehicle and assessing their steering correction. We then used the 3 seconds of neural activity before the unexpected event to derive alpha functional connectivity in the first analysis and alpha power in the second analysis, and we employed linear regression to separately investigate their relationship to 3 metrics of driving performance (lane deviation, reaction time (RT), and heading error). We find that the locations involved in our network analysis also show the strongest modulation of alpha activity. Interestingly, the network pattern suggests a posterior to anterior directionality, consistent with bottom-up theories of attention, and these results may reflect a gain control model of attention in which ongoing attention is modulated through coordinated, network activity. (PsycINFO Database Record

Resting heart rate variability is associated with ex-Gaussian metrics of intra-individual reaction time variability.

The relationships between vagally mediated heart rate variability (vmHRV) and the cognitive mechanisms underlying performance can be elucidated with ex-Gaussian modeling-an approach that quantifies two different forms of intra-individual variability (IIV) in reaction time (RT). To this end, the current study examined relations of resting vmHRV to whole-distribution and ex-Gaussian IIV. Subjects (N = 83) completed a 5-minute baseline while vmHRV (root mean square of successive differences; RMSSD) was measured. Ex-Gaussian (sigma, tau) and whole-distribution (standard deviation) estimates of IIV were derived from reaction times on a Stroop task. Resting vmHRV was found to be inversely related to tau (exponential IIV) but not to sigma (Gaussian IIV) or the whole-distribution standard deviation of RTs. Findings suggest that individuals with high vmHRV can better prevent attentional lapses but not difficulties with motor control. These findings inform the differential relationships of cardiac vagal control to the cognitive processes underlying human performance.

Effect of rest-pause vs. traditional bench press training on muscle strength, electromyography, and lifting volume in randomized trial protocols.

PURPOSE: Rest-pause (4-s unloaded rest between repetitions) training effects on one repetition maximum (1 RM), lifting volume, and neural activation via electromyography (EMG) are currently vague in the literature and can benefit strength and conditioning professionals for resistance training programme design. Therefore, this study compared 1 RM, neural activation via (EMG), and volume differences between rest-pause vs. traditional resistance training. METHODS: Trained males (N = 20) were randomly assigned to either a rest-pause or a traditional training group. Pre- and post-1 RM testing was recorded. Training sessions were completed twice a week for 4 weeks and consisted of four sets of bench press to volitional fatigue at 80% of pre-test 1 RM with a 2-min rest between sets. Total volume completed was recorded on each training day. Neural activation of the pectoralis major was measured on the first and last training days. RESULTS: A two-way repeated-measures ANOVA indicated both groups significantly increased their 1 RMs following the 4-week training protocol (p < .05). However, no significant differences were found in 1 RM and neural activation between the two groups (p > .05). An independent samples t test indicated that total volume lifted was significantly higher for the rest-pause group (56,778 vs. 38,315 lbs; p < .05) throughout the protocol and independently during weeks 2, 3, and 4. CONCLUSIONS: While strength and neural activation changes did not differ between groups, both increased 1 RMs and the rest-pause group achieved greater increases in volume than the traditional group. If volume is the focus of training, the rest-pause method should be utilized.

Estimating direction in brain-behavior interactions: Proactive and reactive brain states in driving.

Conventional neuroimaging analyses have ascribed function to particular brain regions, exploiting the power of the subtraction technique in fMRI and event-related potential analyses in EEG. Moving beyond this convention, many researchers have begun exploring network-based neurodynamics and coordination between brain regions as a function of behavioral parameters or environmental statistics; however, most approaches average evoked activity across the experimental session to study task-dependent networks. Here, we examined on-going oscillatory activity as measured with EEG and use a methodology to estimate directionality in brain-behavior interactions. After source reconstruction, activity within specific frequency bands (delta: 2-3Hz; theta: 4-7Hz; alpha: 8-12Hz; beta: 13-25Hz) in a priori regions of interest was linked to continuous behavioral measurements, and we used a predictive filtering scheme to estimate the asymmetry between brain-to-behavior and behavior-to-brain prediction using a variant of Granger causality. We applied this approach to a simulated driving task and examined directed relationships between brain activity and continuous driving performance (steering behavior or vehicle heading error). Our results indicated that two neuro-behavioral states may be explored with this methodology: a Proactive brain state that actively plans the response to the sensory information and is characterized by delta-beta activity, and a Reactive brain state that processes incoming information and reacts to environmental statistics primarily within the alpha band.