Conservation of the oligomeric state of native VDAC1 in detergent micelles.

The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods (i.e. chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent KD for ATP of 1.2 mM.

Tree phenology and carbon dioxide fluxes - use of digital photography for process-based interpretation at the ecosystem scale

Vegetation phenology is an important indicator of climate change and climate variability and it is strongly connected to biospheric–atmospheric gas exchange. We aimed to evaluate the applicability of phenological information derived from digital imagery for the interpretation of CO2 exchange measurements. For the years 2005–2007 we analyzed seasonal phenological development of 2 temperate mixed forests using tower-based imagery from standard RGB cameras. Phenological information was jointly analyzed with gross primary productivity (GPP) derived from net ecosystem exchange data. Automated image analysis provided reliable information on vegetation developmental stages of beech and ash trees covering all seasons. A phenological index derived from image color values was strongly correlated with GPP, with a significant mean time lag of several days for ash trees and several weeks for beech trees in early summer (May to mid-July). Leaf emergence dates for the dominant tree species partly explained temporal behaviour of spring GPP but were also masked by local meteorological conditions. We conclude that digital cameras at flux measurement sites not only provide an objective measure of the physiological state of a forest canopy at high temporal and spatial resolutions, but also complement CO2 and water exchange measurements, improving our knowledge of ecosystem processes.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Reproducibility of effects of homeopathically potentised gibberellic acid on the growth of Lemna gibba L. in a randomised and blinded bioassay

BACKGROUND: Reproducibility of basic research investigations in homeopathy is challenging. This study investigated if formerly observed effects of homeopathically potentised gibberellic acid (GA3) on growth of duckweed (Lemna gibba L.) were reproducible. METHODS: Duckweed was grown in potencies (14x-30x) of GA3 and one time succussed and unsuccussed water controls. Outcome parameter area-related growth rate was determined by a computerised image analysis system. Three series including five independent blinded and randomised potency experiments (PE) each were carried out. System stability was controlled by three series of five systematic negative control (SNC) experiments. Gibbosity (a specific growth state of L. gibba) was investigated as possibly essential factor for reactivity of L. gibba towards potentised GA3 in one series of potency and SNC experiments, respectively. RESULTS: Only in the third series with gibbous L. gibba L. we observed a significant effect (p = 0.009, F-test) of the homeopathic treatment. However, growth rate increased in contrast to the former study, and most biologically active potency levels differed. Variability in PE was lower than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. CONCLUSIONS: Gibbosity seems to be a necessary condition for reactivity of L. gibba to potentised GA3. Further still unknown conditions seem to govern effect direction and the pattern of active and inactive potency levels. When designing new reproducibility studies, the physiological state of the test organism must be considered. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

Functional wiring of hypocretin and LC-NE neurons: implications for arousal.

To survive in a rapidly changing environment, animals must sense their external world and internal physiological state and properly regulate levels of arousal. Levels of arousal that are abnormally high may result in inefficient use of internal energy stores and unfocused attention to salient environmental stimuli. Alternatively, levels of arousal that are abnormally low may result in the inability to properly seek food, water, sexual partners, and other factors necessary for life. In the brain, neurons that express hypocretin neuropeptides may be uniquely posed to sense the external and internal state of the animal and tune arousal state according to behavioral needs. In recent years, we have applied temporally precise optogenetic techniques to study the role of these neurons and their downstream connections in regulating arousal. In particular, we have found that noradrenergic neurons in the brainstem locus coeruleus (LC) are particularly important for mediating the effects of hypocretin neurons on arousal. Here, we discuss our recent results and consider the implications of the anatomical connectivity of these neurons in regulating the arousal state of an organism across various states of sleep and wakefulness.

Baclofen and gamma-hydroxybutyrate differentially altered behavior, EEG activity and sleep in rats.

OBJECTIVES Animal and human studies have shown that sleep may have an impact on functional recovery after brain damage. Baclofen (Bac) and gamma-hydroxybutyrate (GHB) have been shown to induce physiological sleep in humans, however, their effects in rodents are unclear. The aim of this study is to characterize sleep and electroencelphalogram (EEG) after Bac and GHB administration in rats. We hypothesized that both drugs would induce physiological sleep. METHODS Adult male Sprague-Dawley rats were implanted with EEG/electromyogram (EMG) electrodes for sleep recordings. Bac (10 or 20 mg/kg), GHB (150 or 300 mg/kg) or saline were injected 1 h after light and dark onset to evaluate time of day effect of the drugs. Vigilance states and EEG spectra were quantified. RESULTS Bac and GHB induced a non-physiological state characterized by atypical behavior and an abnormal EEG pattern. After termination of this state, Bac was found to increase the duration of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep (∼90 and 10 min, respectively), reduce sleep fragmentation and affect NREM sleep episode frequency and duration (p<0.05). GHB had no major effect on vigilance states. Bac drastically increased EEG power density in NREM sleep in the frequencies 1.5-6.5 and 9.5-21.5 Hz compared to saline (p<0.05), while GHB enhanced power in the 1-5-Hz frequency band and reduced it in the 7-9-Hz band. Slow-wave activity in NREM sleep was enhanced 1.5-3-fold during the first 1-2 h following termination of the non-physiological state. The magnitude of drug effects was stronger during the dark phase. CONCLUSION While both Bac and GHB induced a non-physiological resting state, only Bac facilitated and consolidated sleep, and promoted EEG delta oscillations thereafter. Hence, Bac can be considered a sleep-promoting drug and its effects on functional recovery after stroke can be evaluated both in humans and rats.

Topographic electrophysiological signatures of FMRI Resting State Networks

Background: fMRI Resting State Networks (RSNs) have gained importance in the present fMRI literature. Although their functional role is unquestioned and their physiological origin is nowadays widely accepted, little is known about their relationship to neuronal activity. The combined recording of EEG and fMRI allows the temporal correlation between fluctuations of the RSNs and the dynamics of EEG spectral amplitudes. So far, only relationships between several EEG frequency bands and some RSNs could be demonstrated, but no study accounted for the spatial distribution of frequency domain EEG. Methodology/Principal Findings: In the present study we report on the topographic association of EEG spectral fluctuations and RSN dynamics using EEG covariance mapping. All RSNs displayed significant covariance maps across a broad EEG frequency range. Cluster analysis of the found covariance maps revealed the common standard EEG frequency bands. We found significant differences between covariance maps of the different RSNs and these differences depended on the frequency band. Conclusions/Significance: Our data supports the physiological and neuronal origin of the RSNs and substantiates the assumption that the standard EEG frequency bands and their topographies can be seen as electrophysiological signatures of underlying distributed neuronal networks.

Time course based artifact identification for independent components of resting-state FMRI

In functional magnetic resonance imaging (fMRI) coherent oscillations of the blood oxygen level-dependent (BOLD) signal can be detected. These arise when brain regions respond to external stimuli or are activated by tasks. The same networks have been characterized during wakeful rest when functional connectivity of the human brain is organized in generic resting-state networks (RSN). Alterations of RSN emerge as neurobiological markers of pathological conditions such as altered mental state. In single-subject fMRI data the coherent components can be identified by blind source separation of the pre-processed BOLD data using spatial independent component analysis (ICA) and related approaches. The resulting maps may represent physiological RSNs or may be due to various artifacts. In this methodological study, we propose a conceptually simple and fully automatic time course based filtering procedure to detect obvious artifacts in the ICA output for resting-state fMRI. The filter is trained on six and tested on 29 healthy subjects, yielding mean filter accuracy, sensitivity and specificity of 0.80, 0.82, and 0.75 in out-of-sample tests. To estimate the impact of clearly artifactual single-subject components on group resting-state studies we analyze unfiltered and filtered output with a second level ICA procedure. Although the automated filter does not reach performance values of visual analysis by human raters, we propose that resting-state compatible analysis of ICA time courses could be very useful to complement the existing map or task/event oriented artifact classification algorithms.

Current state of the art in myocardial tissue engineering

Myocardial tissue engineering aims to repair, replace, and regenerate damaged cardiac tissue using tissue constructs created ex vivo. This approach may one day provide a full treatment for several cardiac disorders, including congenital diseases or ventricular dysfunction after myocardial infarction. Although the ex vivo construction of a myocardium-like tissue is faced with many challenges, it is nevertheless a pressing objective for cardiac reparative medicine. Multidisciplinary efforts have already led to the development of promising viable muscle constructs. In this article, we review the various concepts of cardiac tissue engineering and their specific challenges. We also review the different types of existing biografts and their physiological relevance. Although many investigators have favored cardiomyocytes, we discuss the potential of other clinically relevant cells, as well as the various hypotheses proposed to explain the functional benefit of cell transplantation.

On the modeling of drug induced respiratory depression in the non-steady-state

The ability of anesthetic agents to provide adequate analgesia and sedation is limited by the ventilatory depression associated with overdosing in spontaneously breathing patients. Therefore, quantitation of drug induced ventilatory depression is a pharmacokinetic-pharmacodynamic problem relevant to the practice of anesthesia. Although several studies describe the effect of respiratory depressant drugs on isolated endpoints, an integrated description of drug induced respiratory depression with parameters identifiable from clinically available data is not available. This study proposes a physiological model of CO2 disposition, ventilatory regulation, and the effects of anesthetic agents on the control of breathing. The predictive performance of the model is evaluated through simulations aimed at reproducing experimental observations of drug induced hypercarbia and hypoventilation associated with intravenous administration of a fast-onset, highly potent anesthetic mu agonist (including previously unpublished experimental data determined after administration of 1 mg alfentanil bolus). The proposed model structure has substantial descriptive capability and can provide clinically relevant predictions of respiratory inhibition in the non-steady-state to enhance safety of drug delivery in the anesthetic practice.

Effects of beta-alanine supplementation and interval training on physiological determinants of severe exercise performance.

INTRODUCTION We aimed to manipulate physiological determinants of severe exercise performance. We hypothesized that (1) beta-alanine supplementation would increase intramuscular carnosine and buffering capacity and dampen acidosis during severe cycling, (2) that high-intensity interval training (HIT) would enhance aerobic energy contribution during severe cycling, and (3) that HIT preceded by beta-alanine supplementation would have greater benefits. METHODS Sixteen active men performed incremental cycling tests and 90-s severe (110 % peak power) cycling tests at three time points: before and after oral supplementation with either beta-alanine or placebo, and after an 11-days HIT block (9 sessions, 4 × 4 min), which followed supplementation. Carnosine was assessed via MR spectroscopy. Energy contribution during 90-s severe cycling was estimated from the O2 deficit. Biopsies from m. vastus lateralis were taken before and after the test. RESULTS Beta-alanine increased leg muscle carnosine (32 ± 13 %, d = 3.1). Buffering capacity and incremental cycling were unaffected, but during 90-s severe cycling, beta-alanine increased aerobic energy contribution (1.4 ± 1.3 %, d = 0.5), concurrent with reduced O2 deficit (-5.0 ± 5.0 %, d = 0.6) and muscle lactate accumulation (-23 ± 30 %, d = 0.9), while having no effect on pH. Beta-alanine also enhanced motivation and perceived state during the HIT block. There were no between-group differences in adaptations to the training block, namely increased buffering capacity (+7.9 ± 11.9 %, p = 0.04, d = 0.6, n = 14) and glycogen storage (+30 ± 47 %, p = 0.04, d = 0.5, n = 16). CONCLUSIONS Beta-alanine did not affect buffering considerably, but has beneficial effects on severe exercise metabolism as well as psychological parameters during intense training phases.

Growth at moderately elevated temperature alters the physiological response of the photosynthetic apparatus to heat stress in pea (Pisum sativum L.) leaves

The impact of heat stress on the functioning of the photosynthetic apparatus was examined in pea (Pisum sativum L.) plants grown at control (25 °C; 25 °C-plants) or moderately elevated temperature (35 °C; 35 °C-plants). In both types of plants net photosynthesis (Pn) decreased with increasing leaf temperature (LT) and was more than 80% reduced at 45 °C as compared to 25 °C. In the 25 °C-plants, LTs higher than 40 °C could result in a complete suppression of Pn. Short-term acclimation to heat stress did not alter the temperature response of Pn. Chlorophyll a fluorescence measurements revealed that photosynthetic electron transport (PET) started to decrease when LT increased above 35 °C and that growth at 35 °C improved the thermal stability of the thylakoid membranes. In the 25 °C-plants, but not in the 35 °C-plants, the maximum quantum yield of the photosystem II primary photochemistry, as judged by measuring the Fv/Fm ratio, decreased significantly at LTs higher than 38 °C. A post-illumination heat-induced reduction of the plastoquinone pool was observed in the 25 °C-plants, but not in the 35 °C-plants. Inhibition of Pn by heat stress correlated with a reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Western-blot analysis of Rubisco activase showed that heat stress resulted in a redistribution of activase polypeptides from the soluble to the insoluble fraction of extracts. Heat-dependent inhibition of Pn and PET could be reduced by increasing the intercellular CO2 concentration, but much more effectively so in the 35 °C-plants than in the 25 °C-plants. The 35 °C-plants recovered more efficiently from heat-dependent inhibition of Pn than the 25 °C-plants. The results show that growth at moderately high temperature hardly diminished inhibition of Pn by heat stress that originated from a reversible heat-dependent reduction of the Rubisco activation state. However, by improving the thermal stability of the thylakoid membranes it allowed the photosynthetic apparatus to preserve its functional potential at high LTs, thus minimizing the after-effects of heat stress.

Hypercoagulation and hyperkinetic blood pressure indicative of physiological loss-of-control despite behavioural control in Africans: The SABPA study.

OBJECTIVES A dissociation between behavioural (in-control) and physiological parameters (indicating loss-of-control) is associated with cardiovascular risk in defensive coping (DefS) Africans. We evaluated relationships between DefS, sub-clinical atherosclerosis, low-grade inflammation and hypercoagulation in a bi-ethnic sex cohort. METHODS Black (Africans) and white Africans (Caucasians) (n = 375; aged 44.6 ± 9.7 years) were included. Ambulatory BP, vascular structure (left carotid cross-sectional wall area (L-CSWA) and plaque counts), and markers of coagulation and inflammation were quantified. Ethnicity/coping style interaction was revealed only in DefS participants. RESULTS A hypertensive state, less plaque, low-grade inflammation, and hypercoagulation were more prevalent in DefS Africans (27-84%) than DefS Caucasians (18-41%). Regression analyses demonstrated associations between L-CSWA and 24 hour systolic BP (R(2) = 0.38; β = 0.78; p < 0.05) in DefS African men but not in DefS African women or Caucasians. No associations between L-CSWA and coagulation markers were evident. CONCLUSION Novel findings revealed hypercoagulation, low-grade inflammation and hyperkinetic BP (physiological loss-of-control responses) in DefS African men. Coupled to a self-reported in-control DefS behavioural profile, this reflects dissociation between behaviour and physiology. It may explain changes in vascular structure, increasing cerebrovascular disease risk in a state of hyper-vigilant coping.

Feasibility of resting-state microstates neurofeedback

Despite major progress, currently available treatment options for patients suffering from schizophrenia remain suboptimal. Antipsychotic medication is one such option, and is helpful in acute phases of the disease. However, antipsychotics cause significant side-effects that often require additional medication, and can even trigger the discontinuation of treatment. Taken together, along with the fact that 20-30% of patients are medication-resistant, it is clear that new medical care options should be developed for patients with schizophrenia. Besides medication, an emerging option to treat psychiatric symptoms is through the use of neurofeedback. This technique has proven efficacy for other disorders and, more importantly, has also proven to be feasible in patients with schizophrenia. One of the major advantages of this approach is that it allows for the influence of brain states that otherwise would be inaccessible; i.e. the physiological markers underlying psychotic symptoms. EEG resting-state microstates are a very interesting electrophysiological marker of schizophrenia symptoms. Precisely, a specific class of resting-state microstates, namely microstate class D, has consistently been found to show a temporal shortening in patients with schizophrenia compared to controls, and this shortening is correlated with the presence positive psychotic symptoms. Under the scope of biological psychiatry, appropriate treatment of psychotic symptoms can be expected to modify the underlying physiological markers accompanying behavioral manifestations of a disease. We reason that if abnormal temporal parameters of resting-state microstates seem to be related to positive symptoms in schizophrenia, regulating this EEG feature might be helpful as a treatment for patients. The goal of this thesis was to prove the feasibility of microstate class D contribution self-regulation via neurofeedback. Given that no other study has attempted to regulate microstates via neurofeedback, we first tested its feasibility in a population of healthy subjects. In the first paper we describe the methodological characteristics of the neurofeedback protocol and its implementation. Neurofeedback performance was assessed by means of linear mixed effects modeling, which provided a complete profile of the neurofeedback’s training response within and between-subjects. The protocol included 20 training sessions, and each session contained three conditions: baseline (resting-state) and two active conditions: training (auditory feedback upon self-regulation performance) and transfer (self-regulation with no feedback). With linear modeling we obtained performance indices for each of them as follows: baseline carryover (baseline increments time-dependent) and learning and aptitude for each of the active conditions. Learning refers to the increase/decrease of the microstate class D contribution, time-dependent during each active condition, and aptitude refers to the constant difference of the microstate class D contribution between each active condition and baseline independent of time. The indices provided are discussed in terms of tailoring neurofeedback treatment to individual profiles so that it can be applied in future studies or clinical practice. In our sample of participants, neurofeedback proved feasible, as all participants at least showed positive results in one of the aforementioned learning indices. Furthermore, between-subjects we observed that the contribution of microstate class D across-sessions increased by 0.42% during baseline, 1.93% during training trials, and 1.83% during transfer. This range is expected to be effective in treating psychotic symptoms in patients. In the second paper presented in this thesis, we explored the possible predictors of neurofeedback success among psychological variables measured with questionnaires. An interesting finding was the negative correlation between “motivational incongruence” and some of the neurofeedback performance indices. Even though this finding requires replication, we discuss it in terms of the interfering effects of incompatible psychological processes with neurofeedback training requirements. In the third paper, we present a meta-analysis on all available studies that have related resting-state microstate abnormalities and schizophrenia. We obtained medium effect sizes for two microstate classes, namely C and D. Combining the meta-analysis results with the fact that microstate class D abnormalities are correlated with the presence of positive symptoms in patients with schizophrenia, these results add further support for the training of this precise microstate. Overall, the results obtained in this study encourage the implementation of this protocol in a population of patients with schizophrenia. However, future studies will have to show whether patients will be able to successfully self-regulate the contribution of microstate class D and, if so, whether this regulation will have an impact on symptomatology.