Search

The acute and long-term consequences of perinatal asphyxia have been extensively investigated, but only a few studies have focused on postnatal asphyxia. In particular, electrophysiological changes induced in the motor cortex by postnatal asphyxia have not been examined so far, despite the critical involvement of this cortical area in epilepsy. In this study, we exposed primary motor cortex slices obtained from infant rats in an age window (16–18 day-old) characterized by high incidence of hypoxia-induced seizures associated with epileptiform motor behavior to 10 min of hypoxia. Extracellular field potentials evoked by horizontal pathway stimulation were recorded in layers II/III of the primary motor cortex before, during, and after the hypoxic event. The results show that hypoxia reversibly depressed glutamatergic synaptic transmission and neuronal excitability. Data obtained in the presence of specific blockers suggest that synaptic depression was mediated by adenosine acting on pre-synaptic A1 receptors to decrease glutamate release, and by a nitric oxide (NO)/cGMP postsynaptic pathway. These effects are neuroprotective because they limit energy failure. The present findings may be helpful in the preclinical search for therapeutic strategies aimed at preventing acute and long-term neurological consequences of postnatal asphyxia.

Cellular contacts modify the way cells migrate in a cohesive group with respect to a free single cell. The resulting motion is persistent and correlated, with cells’ velocities self-aligning in time. The presence of a dense agglomerate of cells makes the application of single particle tracking techniques to define cells dynamics difficult, especially in the case of phase contrast images. Here, we propose an original pipeline for the analysis of phase contrast images of the wound healing scratch assay acquired in time-lapse, with the aim of extracting single particle trajectories describing the dynamics of the wound closure. In such an approach, the membrane of the cells at the border of the wound is taken as a unicum, i.e., the wound edge, and the dynamics is described by the stochastic motion of an ensemble of points on such a membrane, i.e., pseudo-particles. For each single frame, the pipeline of analysis includes: first, a texture classification for separating the background from the cells and for identifying the wound edge; second, the computation of the coordinates of the ensemble of pseudo-particles, chosen to be uniformly distributed along the length of the wound edge. We show the results of this method applied to a glioma cell line (T98G) performing a wound healing scratch assay without external stimuli. We discuss the efficiency of the method to assess cell motility and possible applications to other experimental layouts, such as single cell motion. The pipeline is developed in the Python language and is available upon request., Basque Government BERC 2018– 2021 Spanish Ministry of Economy and Competitiveness MINECO via the BCAM Severo Ochoa SEV-2017-0718 accreditation

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder wherein symptoms resembling aspects of aging are manifested at a very early age. It is a genetic condition that occurs due to a de novo mutation in the LMNA gene encoding for the nuclear structural protein lamin A. The lamin family of proteins are thought to be involved in nuclear stability, chromatin structure and gene expression and this leads to heavy effects on the regulation and functionality of the cell machinery. The functional role of the large-conductance calcium-activated potassium channels (BKCa) is still unclear, but has been recently described a strong relationship with their membrane expression, progerin nuclear levels and the ageing process. In this study, we found that: i) the outward potassium membrane current amplitude and the fluorescence intensity of the BKCa channel probe showed higher values in human dermal fibroblast obtained from patients affected by HGPS if compared to that from healthy young subjects; ii) this result appears to correlate with a basic cellular activity such as the replicative boost. We suggest that studying the HGPS also from the electrophysiological point of view might reveal new clues about the normal process of aging.

none 9 no In a Plasma Focus device the plasma collapses into the pinch where it reaches thermonuclear conditions for a few tens of nanoseconds, becoming a multi-radiation source. The nature of the radiation generated depends on the gas filling the chamber and the device working parameters. The self-collimated electron beam generated in the backward direction with respect to the plasma motion is one of the main radiation sources of interest also for medical applications. The electron beam may be guided against a high Z material target to produce an X-ray beam. This technique offers an ultra-high dose rate source of X-rays, able to deliver during the pinch a massive dose (up to 1 Gy per discharge for the PFMA-3 test device), as measured with EBT3 GafchromicⒸfilm tissue equivalent dosimeters. Given the stochastic behavior of the discharge process, a reliable on-line estimate of the dose-delivered is a very challenging task, in some way preventing a systematic application as a potentially interesting therapy device. This work presents an approach to linking the dose registered by the EBT3 GafchromicⒸfilms with the information contained in the signal recorded during the current discharge process. Processing the signal with the Wigner-Ville distribution, a spectrogram was obtained, displaying the information on intensity at various frequency scales, identifying the band of frequencies representative of the pinch events and define some patterns correlated with the dose. Sumini, M.; Mostacci, D.; Tartari, A.; Mazza, A.; Cucchi, G.; Isolan, L.; Buontempo, F.; Zironi, I.; Castellani, G. Sumini, M.; Mostacci, D.; Tartari, A.; Mazza, A.; Cucchi, G.; Isolan, L.; Buontempo, F.; Zironi, I.; Castellani, G.

PEDOT:PSS is a highly conductive material with good thermal and chemical stability and enhanced biocompatibility that make it suitable for bioengineering applications. The electrical control of the oxidation state of PEDOT:PSS films allows modulation of peculiar physical and chemical properties of the material, such as topography, wettability, and conductivity, and thus offers a possible route for controlling cellular behavior. Through the use of (i) the electrophysiological response of the plasma membrane as a biosensor of the ionic availability; (ii) relative abundance around the cells via Xray spectroscopy; and (iii) atomic force microscopy to monitor PEDOT:PSS film thickness relative to its oxidation state, we demonstrate that redox processes confer to PEDOT:PSS the property to modify the ionic environment at the film−liquid interface through a “sponge-like” effect on ions. Finally, we show how this property offers the capability to electrically control central cellular properties such as viability, substrate adhesion, and growth, paving the way for novel bioelectronics and biotechnological applications.

[This corrects the article DOI: 10.1371/journal.pone.0199312.].

Conducting polymers are promising materials for tissue engineering applications, since they can both provide a biocompatible scaffold for physical support of living cells, and transmit electrical and mechanical stimuli thanks to their electrical conductivity and reversible doping. In this work, thin films of one of the most promising materials for bioelectronics applications, poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS), are prepared using two different techniques, spin coating and electrochemical polymerization, and their oxidation state is subsequently changed electrochemically with the application of an external bias. The electrochemical properties of these different types of PEDOT:PSS are studied through cyclic voltammetry and spectrophotometry to assess the effectiveness of the oxidation process and its stability over time. Their surface physical properties and their dependence on the redox state of PEDOT:PSS are investigated using atomic force microscopy (AFM), water contact angle goniometry and sheet resistance measurements. Finally, human glioblastoma multiforme cells (T98G) and primary human dermal fibroblasts (hDF) are cultured on PEDOT:PSS films with different oxidation states, finding that the effect of the substrate on the cell growth rate is strongly cell-dependent: T98G growth is enhanced by the reduced samples, while hDF growth is more effective only on the oxidized substrates that show a strong chemical interaction with the cell culture medium.

Nuclear magnetic resonance (NMR) techniques, in particular 1H relaxometry, enable the acquisition of information on the water exchange between different compartments in real-time and in a non-invasive and non-destructive way. Therefore, these are currently the methods of choice for studying the diffusion of water in living tissues and cells. The multi-compartmental nature of these systems may generate multi-exponential decays of the nuclear magnetization vector components to be analyzed in terms of discrete or continuous distributions of the so-called relaxation times. We have evaluated the sensitivity of the continuous distributions of the transverse component relaxation time (T 2) to detect and monitor, over time, the water diffusion caused by osmotic pressure variations in tumor cells suspended in isotonic and hypotonic/hypertonic culture media. Our results indicated that the T 2 distribution shapes were capable of describing the water compartmentalization and exchange, with evolutions that strongly depended on the degree of tonicity alterations. In order to validate the NMR results we also monitored the samples by phase contrast optical microscopy finding consistency between the two methods. Moreover, a study of T 2-T 2 correlation function allowed us to observe the exchange between cytoplasmic water and intracellular 1H spin groups. Using a two-site exchange model we also discussed, as a function of the uncertainties on the intra-to-extra water fraction and on the T 2 values, the computed intracellular average water lifetime at equilibrium, an important parameter recently proposed as indicator of tumor aggressiveness. The results support the idea that the information conveyed by the T 2 parameter might be useful for an MRI diagnostic approach to diseases in which the fluid tonicity or the cellular membranes permeability are altered.

Despite the experimental evidence of significant biological effects of extremely low frequency (ELF) magnetic fields (MFs), the underlying mechanisms are still unclear. Among the few mechanisms proposed, of particular interest is the so called "ion parametric resonance (IPR)" hypothesis, frequently referred to as theoretical support for medical applications. We studied the effect of different combinations of static (DC) and alternating (AC) ELF MFs tuned on resonance conditions for potassium (K(+)) on TEA-sensitive voltage-dependent outward K(+) currents in the human neuroblastoma BE(2)C cell line. Currents through the cell membrane were measured by whole-cell patch clamp before, during, and after exposure to MF. No significant changes in K(+) current density were found. This study does not confirm the IPR hypothesis at the level of TEA-sensitive voltage-dependent outward K(+) currents in our experimental conditions. However, this is not a direct disprove of the hypothesis, which should be investigated on other ion channels and at single channel levels also.

In biological applications, conjugated polymers offer many advantages compared to inorganic semiconductors, due to their favorable electrical properties and their biocompatibility. Many different parameters affect the cell-substrate interaction and in this work we focus our attention on the role played by the oxidation state and surface morphology of conducting polymer substrates. We realized cell culture substrates using a thin film of a biocompatible conducting polymer widely employed in organic electronics, poly(3,4-ethylene dioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). The oxidation state of the samples was electrochemically modified through the application of a fixed potential, and they were subsequently characterized by atomic force microscopy and optical spectroscopy. Using these techniques we have been able to measure the oxidation state of the polymer films, and to asses that its surface roughness does not depend on its oxidation state. Furthermore, human dermal fibroblast (hDF) were grown on PEDOT:PSS films with different oxidation state, in order to test their efficacy as cell culture substrates and their biocompatibility.

A pulsed plasma device “Mather type” Plasma Focus (PFMA-3) has been recently put into operation for a possible application to radiotherapy treatment of malignant cells. Some preliminary data about the better efficiency of PFMA-3 to induce cell damage compared to conventional X-ray devices (XRT) have already been obtained in breast adenocarcinoma cell line MCF-7. To better assess the advantage of the very high dose rate delivered by PFMA-3, we explored its biological effectiveness on a human melanoma cell line SK-Mel28, well known for its radioresistance, in comparison with conventional sources.

Aging is a complex process resulting from, among other, dynamic non-linear interactions between genetics and environment. Centenarians are the best example of successful aging in humans, as they escaped from, or largely postponed, major age-related diseases. Ionic fluxes changes play a key role in several patho-physiological cellular processes, but their relation to human aging is largely unexplored. In the present study we have compared patch-clamp potassium (K+) current recordings from dermal fibroblasts (DF) obtained from young, elderly and centenarian donors. We found that in DF from elderly donors, but not from centenarians, K+ current amplitude is significantly smaller with respect to DF from young donors. Moreover, cell membrane capacitance of DF from elderly donors is smaller with respect to young donors and centenarians. We also observed that the voltage-gated Shaker Kv1.1 channel is expressed in higher percentage of elderly's and centenarian's DF than young's, whereas the large-conductance calcium-activated K+ (BKCa) channel beta 1 subunit is expressed in lower percentage of centenarian's DF than in elderly's and young's. The maintenance of "young" K+ currents and the peculiar age-related remodeling of K+ channel subtypes in centenarian's DF is likely associated with successful aging and might provide a predictive marker of longevity. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Radiation therapy is a useful and standard tumor treatment strategy. Despite recent advances in delivery of ionizing radiation, survival rates for some cancer patients are still low because of recurrence and radioresistance. This is why many novel approaches have been explored to improve radiotherapy outcome. Some strategies are focused on enhancement of accuracy in ionizing radiation delivery and on the generation of greater radiation beams, for example with a higher dose rate. In the present study we proposed an in vitro research of the biological effects of very high dose rate beam on SK-Mel28 and A375, two radioresistant human melanoma cell lines. The beam was delivered by a pulsed plasma device, a “Mather type” Plasma Focus for medical applications. We hypothesized that this pulsed X-rays generator is significantly more effective to impair melanoma cells survival compared to conventional X-ray tube. Very high dose rate treatments were able to reduce clonogenic efficiency of SK-Mel28 and A375 more than the X-ray tube and to induce a greater, less easy-to-repair DNA double-strand breaks. Very little is known about biological consequences of such dose rate. Our characterization is preliminary but is the first step toward future clinical considerations.

MARK-AGE aims at the identification of biomarkers of human aging capable of discriminating between the chronological age and the effective functional status of the organism. To achieve this, given the structure of the collected data, a proper statistical analysis has to be performed, as the structure of the data are non trivial and the number of features under study is near to the number of subjects used, requiring special care to avoid overfitting. Here we described some of the possible strategies suitable for this analysis. We also include a description of the main techniques used, to explain and justify the selected strategies. Among other possibilities, we suggest to model and analyze the data with a three step strategy

The quasi-continuous distributions of spin-lattice (T 1) and spin-spin (T 2) relaxation times of a population of cells kept under stress conditions for about 400 h has been obtained by 1H-NMR relaxometry. The comparison with the results obtained from the analysis of cell viability and morphological variations by phase contrast optical microscopy and performed with acquisition times, duration and conditions matching those of the relaxometry experiments allowed us to identify three main phases of the processes related to the exchanges of water and remodeling of cellular compartments: between 0 and 40 h the intra- and extra-cellular compartments are characterized by a T 2 of ~250 ms and of ~2 s respectively and the cell population half-life has been experimentally evaluated to be ~45 h; after 40 and until 100 h cells NMR parameter changes suggest that the confined water is increasing according to the progressive fragmentation of the cells membrane; over 100 h almost all cells are non-viable and the constancy of the main NMR parameters reflects the reaching of the final equilibrium of the system. In conclusion our observations validated the use of NMR as a non-destructive, non-invasive powerful technique for monitoring the progression of cellular processes involving compartments water exchange and reorganization.

A comparative study has been performed on the effects of high-dose-rate (DR) X-ray beams produced by a plasma focus device (PFMA-3), to exploit its potential medical applications (e.g. radiotherapy), and low-DR X-ray beams produced by a conventional source (XRT). Experiments have been performed at 0.5 and 2 Gy doses on a human glioblastoma cell line (T98G). Cell proliferation rate and potassium outward currents (IK) have been investigated by time lapse imaging and patch clamp recordings. The results showed that PFMA-3 irradiation has a greater capability to reduce the proliferation rate activity with respect to XRT, while it does not affect IK of T98G cells at any of the dose levels tested. XRT irradiation significantly reduces the mean IK amplitude of T98G cells only at 0.5 Gy. This work confirms that the DR, and therefore the source of radiation, is crucial for the planning and optimisation of radiotherapy applications. © The Author 2015.

Galvanotaxis is a bioelectronic phenomenon described since the end of the 19th century, that indicates the movement of a living organism in response to an electrical stimulus. At the cellular level, this particular behavior seems to be present in many physiological processes such as embryogenesis, angiogenesis and tumor metastases. In vitro studies clearly showed that some types of cells, when subjected to electric fields of the same magnitude than the endogenous ones, tend to orientate and to migrate along the axes of the electrical field, targeting the cathode or to the anode depending on the cell type. The main objective of this work is the design and development of an electronic system capable to create and control galvanotactic events. We also performed some preliminary experiments finalized to test whether this device allows reproducing the galvanotaxis phenomena in a scientific and easily reproducible manner.

It has previously been proposed that prefrontal cortex may have some role in keeping temporal cortex-based representations 'on-line' during a working memory task. To test this hypothesis, the effects of electrolytic prefrontal cortex lesions on the firing of area TE and perirhinal cortex (PRC) neurons were examined while rats performed a delayed non-match to position task in the T-maze. The behavioural performance of control (n = 4) and lesioned (n = 4) animals were similar during this task, and many neurons displayed a statistically significant location-related variation in firing rate during the sample (44/56 neurons) and test (39/56 neurons) phases. Units from prefrontal-lesioned animals (82%) were more likely to display a significant variation in firing across the maze compared to controls (50%; P < 0.01), and to have more discrete location-related properties (50% of neurons) compared to the control (5%) group (P < 0.0005). This finding suggests that prefrontal cortex normally modulates the transmission and/or processing of spatial information in area TE/PRC during a working memory task. Modulation could be mediated through direct connections between the structures or via prefrontal control of subcortical structures. This finding has implications for our understanding of prefrontal-temporal involvement in memory and cognitive disorders.

Despite the experimental evidence of significant biological effects of extremely low frequency (ELF) magnetic fields (MFs), the underlying mechanisms are still unclear. Among the few mechanisms proposed, of particular interest is the so called "ion parametric resonance (IPR)" hypothesis, frequently referred to as theoretical support for medical applications. We studied the effect of different combinations of static (DC) and alternating (AC) ELF MFs tuned on resonance conditions for potassium (K(+)) on TEA-sensitive voltage-dependent outward K(+) currents in the human neuroblastoma BE(2)C cell line. Currents through the cell membrane were measured by whole-cell patch clamp before, during, and after exposure to MF. No significant changes in K(+) current density were found. This study does not confirm the IPR hypothesis at the level of TEA-sensitive voltage-dependent outward K(+) currents in our experimental conditions. However, this is not a direct disprove of the hypothesis, which should be investigated on other ion channels and at single channel levels also.

The telencephalo-habenulo-interpeduncular system has been anatomically and neurochemically characterized in mammals. However, little is known about these important forebrain-midbrain connections in non-mammalian vertebrates, although available data suggest that they are evolutionarily relatively conservative. Previous ultrastructural studies in the goldfish confirmed the presence of massive telencephalohabenular and habenulointerpeduncular projections and demonstrated a minor direct telencephalointerpeduncular connection. Here we report the anterograde and retrograde transport of lipophilic fluorescent carbocyanine dye from the interpeduncular nucleus and the habenular nuclei in the fixed goldfish brain. The application of dye into the interpeduncular nucleus resulted in massive labeling of the fasciculus retroflexus and of the habenular neurons. A few scattered neurons were also seen in the dorsal nucleus of area ventralis telencephali. Application of dye into the habenulae resulted in anterograde transport through the medial and lateral olfactory tracts to some cell bodies in the anterior and posterior zone of area ventralis telencephali and in perikarya of the bed nucleus and in the entopeduncular nucleus. These results demonstrate the origin of the direct telencephalointerpeduncular projection in the goldfish and confirm some important homologies with forebrain-midbrain projections in land vertebrates.

The habenular nuclei are the major sources of projections to the interpeduncular nucleus. The habenular neurons of the goldfish are ChAT and NADPH-diaphorase positive. The localization of these two enzymes in the habenulae suggests the involvement of acetylcholine and nitric oxide (the product of NADPH-diaphorase activity in the nervous tissue) in the habenulo-interpeduncular connection. This finding is supported by the presence of ChAT and NADPH-diaphorase activity in the neuropilar area of the interpeduncular nucleus. This activity was depleted 12 days after habenular ablation. The overlap of ChAT and NADPH-diaphorase localization in goldfish habenular neurons is consistent with previous observations on the co-localization of these two enzymes in some brainstem neurons of other vertebrates. It is, however, not identical to the localization of NADPH-diaphorase in the habenulae and in the interpeduncular nucleus of the rat.

Surgical ablations of telencephalon or telencephalon plus habenular nuclei (HBN) have been performed for studying anatomical connections of the telencephalo-habenulo- interpeduncular system of the goldfish. The results of the ultrastructural studies confirmed the presence of massive telencephalo-habenular and habenulo-interpeduncular projections and, in addition, demonstrated a minor direct telencephalo-interpeduncular connection. Immunohistochemical localization of substance P in the interpeduncular nucleus (IPN) after telencephalic ablation failed to demonstrate the involvement of this neurotransmitter in the direct telencephalo-interpeduncular projection, while telencephalon plus HBN ablation reduced substance P immunoreactivity in the IPN. The different distribution of degenerating terminals converging on the IPN from the HBN and from the telencephalon suggested a subnuclear organization of this area, as described in mammals.

Human aging is associated with complex alterations that contribute to remodelling of physiological processes and ultimately manifests in loss of tissue/organ function. Peripheral blood T cells do not escape this phenomenon and undergo profound remodelling with aging. Thus, investigating the effects of aging on T cells transcriptomics and identifying the underlying regulatory mechanisms can be of extreme importance to understand the aging process in the Immune System (IS). To this aim, we performed an analysis of gene expression data of T cells collected from peripheral blood of 25 healthy human donors of different age from 25 to more than 95 years, in order to characterize changes that occur throughout the entire adult lifespan. By means of microarray analysis, we observed large groups of genes exhibiting non-monotonic expression patterns over time: such behaviour, that could not be observed in typical “two-group” experiments (e.g. young vs. old people) highlights similarities in gene expression profiles of young and “successfully aged” individuals. Genes whose expression profiles change during lifespan were grouped into three main patterns (eigenmodes) to which different biological functions were significantly associated. The analysis of KEGG pathways to which these genes belong indicated that the biological processes altered in T cell aging are not only those typically associated with immune cells (Jak–STAT signalling, T cell receptor signalling, cytokine–cytokine receptor interactions, etc.) but also some not specific of immune cells, such as long-term depression, PPAR and mTOR signalling, glucose and glutathione metabolism, suggesting that T cell aging may be representative of a more generalised aging phenomenon. Thus, the T cell may represent a useful cellular model to study organismal aging. We further searched for over-represented transcription factor binding sites (TFBSs) in the promoter regions of genes clustered by similarity of their age-related patterns to evidence possible co-regulation. A comparison between over-representation of TFBSs and the time course of the corresponding transcription factor (TF) expression levels revealed that a restricted group of TFs may play a central role in driving aging-specific changes in gene expression of T cells.

Synaptic plasticity is the process by which neurons change the efficacy (or the strength) of their connections (synapses). In the connectionist paradigm, synaptic plasticity is a central concept because it is widely accepted that memory and learning are biologically encoded by variations of neuronal connections strength. In a more general sense, activity-dependent synaptic plasticity is assumed to be necessary and sufficient to encode and store memory in specific brain areas. Another feature of synaptic plasticity is the bidirectionality, which is the capability to increase or decrease the synaptic weights, thus encompassing the classical Hebbian paradigm.

The environment in which alcohol consumption occurs may trigger later relapse in alcohol abusers. In this study we tested whether an alcohol-associated environment would induce alcohol-seeking behavior. Male rats (n=8) were trained to lever press for oral alcohol reinforcement in a distinctive context. Animals were water-restricted to encourage high ethanol intakes, to determine if the context may influence relapse at levels of ethanol consumption that are likely to alter hippocampal spatial functions. Responding was then extinguished in a different context. Placement of the rats back into the original context in which they self-administered alcohol induced a significant increase in lever press responding as compared to extinction levels of responding. The ability of the alcohol context to support alcohol-seeking behavior was maintained over three weeks, with no significant diminution, suggesting that regular consumption of moderate doses of ethanol during training (1.31 0.07 SEM g/kg per 1-hr session consumed on average over 15-20 days) did not affect the formation of the context-ethanol association. A second group of rats (n=6) was trained to lever press for sucrose reinforcement; this group also demonstrated context-dependent reinstatement, although the degree of reinstatement decreased over repeated tests, returning to extinction values after three weeks. These findings indicate that contextual conditioning has a long-term impact on ethanol-seeking behavior.

We characterize different tumour types in search for multi-tumour drug targets, in particular aiming for drug repurposing and novel drug combinations. Starting from 11 tumour types from The Cancer Genome Atlas, we obtain three clusters based on transcriptomic correlation profiles. A network-based analysis, integrating gene expression profiles and protein interactions of cancer-related genes, allows us to define three cluster-specific signatures, with genes belonging to NF-κB signaling, chromosomal instability, ubiquitin-proteasome system, DNA metabolism, and apoptosis biological processes. These signatures have been characterized by different approaches based on mutational, pharmacological and clinical evidences, demonstrating the validity of our selection. Moreover, we define new pharmacological strategies validated by in vitro experiments that show inhibition of cell growth in two tumour cell lines, with significant synergistic effect. Our study thus provides a list of genes and pathways that could possibly be used, singularly or in combination, for the design of novel treatment strategies., Tumours of different tissues can show similarities in genomic alterations. Here, the authors combine tumour transcriptome and protein interaction data in a network-based analysis of 11 tumours types, and identify clusters of tumours with specific signatures for multi-tumour drug targeting and survival prognosis.