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In this study we argue that the appropriateness of an answer to a why-question, potentially bearing on multiple contrast classes, is mainly influenced by the focalized argument, which identifies the relevant reference set. The focalization structure, however, interacts in a non-trivial way with the thematic structure and its accessibility, suggesting a general (independent) prominence of the direct object (DO) over the indirect one (IO). In correlation to that, we also observed that DO appears more resistant to extraction compared to IO, while it seems felicitous in a post-IO focalized low position (light NP-shifting). These contrasts are obtained by running five distinct experiments in Italian targeting various dislocation configurations: Four forced choice tasks manipulating leftward dislocation (i) clefting versus (ii) fronting versus (iii) clitic left dislocation and (iv) postverbal reordering (canonical DO IO order vs. IO DO) in ditransitive predicates. Then (v) an acceptability judgment study was administered to assess the difficulty in figuring out a licensing context coherent with the argument ordering, provided in the why-question. To minimize the interacting factors, all sentences included null subjects and a context was provided for each experimental item in the forced choice tasks, introducing the relevant contrast classes.

Introduction Telemedicine is the exchange of medical information from one site to another via electronic communications with the goal of improving a patient's clinical health status. Prior studies have identified the absence of a standardized assessment tool for evaluating telemedicine encounters. This study aims to collect and to analyze questionnaires used for the assessment of audiovisual telemedicine encounters from a patient perspective and aims to identify reasons driving the use of self-developed questionnaires. Methods We conducted a systematic search in PubMed for studies that used survey questionnaires to assess synchronous audiovisual telemedicine encounters from 2016 to 2021. We categorized questionnaires used into validated and non-validated types, and for each of them, collected questions, response format, author, year, specialty, and country of publication. Results and Discussion We analyzed a total of 71 articles. We found that only 16 studies used three validated questionnaires. The remaining 55 studies used non-validated questionnaires. Non-validated questionnaires had a high variability in length and used Likert scales, binary responses, multiple choice, and open-ended answers. We found only eight studies in which the authors gave a reason for resorting to designing their own questionnaires. This review reveals insufficient standardized survey questionnaires to be used for the assessment of audiovisual telemedicine encounters. Future research initiatives should focus on developing a standardized and validated instrument well accepted by researchers.

Business incubators are organisations that support the growth of small companies, including start-ups, by providing various resources and services. The aims of this article were to assess the characteristics of business incubators in non-hub cities located in Georgia and South Carolina and to describe the major differences between incubators located in non-hub and hub cities. We surveyed 5 non-hub incubators and visited and analysed qualitatively 10 incubators, 5 in hub cities and 5 in non-hub cities. Results showed that incubators in non-hub cities have less focus and less access to funding capital compared with incubators in hub cities. The implementation of a mesh network among incubators in non-hub cities may help sharing resources, know-how, talents and investments with the goal of being able to compete with incubators in hub cities. While currently incubators in non-hub cities cannot offer the same services to their members, they can still play an important role in giving the people in their communities an opportunity to start a new business, find jobs and increase their income. Business incubators in non-hub cities can ultimately positively impact the overall quality of life of the population they serve. Finally, we proposed that a focus on public health innovation may help incubators in non-hub cities to be successful.

[EN] Anthropogenic activities are causing increased noise levels in the marine environment. To date, few studies have been undertaken to investigate the effects of different noise frequencies on the behaviour of juvenile fish. In this study, the behavioural changes of juvenile gilthead seabream (Sparus aurata) are evaluated when exposed to white noise filtered in third-octave bands centred at 63, 125, 500, and 1000 Hz (sound pressure level, 140-150 dB re 1 mu& x3a1;a) for 7 h. The group dispersion, motility, and swimming height of the fish were analysed before and during the acoustic emission. Dispersion of the fish was found to reduce immediately upon application of low frequency sound (63 and 125 Hz) with a return to control condition after 2 h (indicative of habituation), whereas at 1 kHz, dispersion increased after 2 h without any habituation. The motility decreased significantly at 63 Hz throughout the 7 h of sound exposure. The swimming height decreased significantly for all frequencies other than 125 Hz. The results of this study highlight significant variations in the behavioural responses of juvenile fish that could have consequences on their fitness and survival., This study was conducted in the framework of the Ph.D. program in the Mediterranean Biodiversity XXXII cycle (International) of the University of Palermo. I.P.-A., M.B.-C. and V.E. acknowledge the financial support of the European Comission-Project No. 11.0661/2018794607/SUB/ENV.C2, risk-based approaches to good environmental status (RAGES). The other founding support comes from the Ph.D. Innovative with Industrial Characterization Programma Operativo Nazionale (PON) 2014-2020 and the projects bilateral Research laboratory on marine and maritime Science Italy-Argentina (CAIMAR) Joint Laboratory Italy-Argentina (Laboratori Congiunti Bilaterali Internazionali of the Italian National Reseach Council, 2017-2019). Document

Introduction The use of telemedicine has broadened as technology that both restores continuity of care during disruptions in healthcare delivery and routinely provides primary care alone or in combination with in-person care. During the Covid-19 outbreak, the use of telemedicine as a routine care modality further accelerated. Methods A review of scientific studies that used telemedicine to provide care from December 2019 to December 2020 is presented. From an initial set of 2,191 articles, 36 studies are analyzed. Evidence is organized and evaluated according to the country of study, the clinical specialty, the technology platform used, and satisfaction and utilization outcomes. Results Thirty-one studies reported high patient satisfaction scores. Eight studies reported satisfaction from both providers and patients with no uniformly accepted assessment instrument. Eight studies conducted a descriptive analysis of telemedicine use and patient adoption patterns. Less than one-third of studies were controlled before/after studies. Most studies were conducted in the USA followed by Europe. Conclusions Reported satisfaction rates are high, consistent with previously documented research, whereas utilization rates increased significantly compared with the prepandemic period. Future work in developing standardized uniform assessment instruments, embedded with each telemedicine system, would increase versatility and agility in the assessment, boosting statistical power and the interpretation of results.

Underwater noise is mainly produced by anthropogenic activities and has increased in recent years. The Marine Strategy Framework Directive aims to monitor “continuous low frequency sound” trends in ambient noise in particular at 1/3rd octave bands centered at 63 and 125 Hz. The aim of this work is to evaluate the possibility of using the results of Sparus aurata juvenile’s behavior for parameterizing a numerical model of the fish behavior. The fishes were stressed with white noise filtered with a band pass filter at 1/3 octave band centered respectively at 63 Hz, 125 Hz, 500 Hz, and 1 kHz (SPL: 140-150 dB re 1µPa). We performed three test replicas for the control (without any sound emission) and for each of the four frequencies. Each trial lasted 7:15 hours in total and included the following three periods in which two video cameras recorded: 30 min before the acoustic emission; 60 min in which the acoustic stress was dispensed; 6 hours after the acoustic emission divided into 6 periods of 1 hour. To obtain the behavioral data (cohesion, motility, swimming height) the fishes were video monitored 15 min before the sound exposure, one hour during the sound exposure and the final 15 minutes of each hour after the sound exposure. The bottom of the tank was divided into squares. Cohesion was evaluated by counting the number of squares occupied by the group; motility by counting the number of squares crossed by each fish; swimming height by counting the fish presence in three zones (deepest, intermediate, and highest). Using Kruskal-Wallis tests and multiple comparisons post-hoc tests we assessed different behaviors of the fishes reacting to different acoustic frequencies. Here, we propose the use of part of these data (the first hour of acoustic emission) for the parameterization of a numerical model using an adaptation of the Boids method. In the model the cohesion, motility and the swimming height are analyzed in the same way as they are analyzed for in vivo video recordings. Preliminary results showed that it is possible to reproduce in the new model the same qualitative behavior of the in vivo recordings with fish. In this way, it will be possible to understand the behavioural responses of fish in laboratory experiments and contribute to the prediction of the impacts of other possible acoustic sounds emitted by human activities through the application of the created numerical model.

Marine life is extremely sensitive to the effects of environmental noise due to its reliance on underwater sounds for basic life functions, such as searching for food and mating. However, the effects on invertebrate species are not yet fully understood. The aim of this study was to determine the biochemical responses of Arbacia lixula exposed to high-frequency noise. Protein concentration, enzyme activity (esterase, phosphatase and peroxidase) and cytotoxicity in coelomic fluid were compared in individuals exposed for three hours to consecutive linear sweeps of 100 to 200 kHz lasting 1 s, and control specimens. Sound pressure levels ranged between 145 and 160 dB re 1μPa. Coelomic fluid was extracted and the gene and protein expression of HSP70 with RT-PCR was evaluated on coelomocytes. A significant change was found in enzyme activity and in the expression of the HSP70 gene and protein compared to the control. These results suggested that high-frequency stimuli elicit a noise-induced physiological stress response in A. lixula, confirming the vulnerability of this species to acoustic exposure. Furthermore, these findings provide the first evidence that cell-free coelomic fluid can be used as a signal to evaluate noise exposure in marine invertebrates.

Anthropic noise is considered a real pollutant, in particular the submarine noise. The impact on biodiversity is not yet sufficiently understood. Further research is needed to evaluate any negative effects. The noises associated with anthropogenic activities are increasing: shipments, seismic surveys, sonar, recreational rowing and future mineral extraction activities from ocean depths (DSM). These noises are having an impact on the welfare of many marine species. The understanding of the effects on biodiversity could be important for the creation of guidelines, laws or rules for the most environmentally sustainable exploitation of natural resources. Our study aims to investigate the motility, biochemical and molecular responses of Arbacia lixula exposed to an acoustic stimulus produced by anthropogenic activities and perhaps perceptible by invertebrates. The animals were divided into a control tank and experimental tank. The specimens were exposed to sonic stress for 3 hours after which biological sampling was performed. The sonic stress used was a linear sweep from 100 to 200 kHz emitted every 1 second. The Sound Pressure Level ranges between 173 and 181 dBrms (re 1μPa), with a peak at 150 kHz. To measure the motility, on the top of the tanks two cameras were placed to make photo each 8 seconds during acoustic stimulus. The bottom of the tanks were divided virtually by squares and the vertical walls were divided by two quote. At each photo we assessed the position of the animals and count the number of squares/levels crossed comparing to the precedent photo. In this way we obtained the motility of the specimens in the three directions (cm/s). After three hours of stimulus projection, the sea urchins were captured and the coelomatic fluid was extracted. The protein concentration and the enzymatic activities of esterase, phosphatase, catalase and peroxidase were measured on the celomocytes and on the supernatant of the celomatic fluid. The gene expression of HSP70 and HSP90 with RT-PCR was evaluated on celomocytes. Exposure to this type of noise produced a significant changing in motility and an increase in the expression of HSPs gene, more so for HSP70. No statistical difference was observed in the extent of enzymatic activities and protein concentration. The results obtained indicate that this type of acoustic stimulus has effects on the behavior and on the gene expression of HSPs of individuals of Arbacia lixula.

The earth’ resources are running out, the population will increase and further sources will be needed. These, were found in the deep ocean. To date the mining activities (Deep Sea Mining, DSM) they have not started. One impact of DSM could be underwater noise. Considering the noise frequencies of anthropic activities in the ocean, and the European directive on underwater noise control, we stressed the animals with four acoustic 1/3 band noises around: 63 Hz, 125 Hz, 500 Hz, 1kHz. We study the effects on Sparus aurata juveniles, and on Mytilus galloprovincialis. We will analyse behavioural., molecular and biochemical responses Los recursos de la tierra se están agotando, la población aumentará y se necesitarán más fuentes. Esaos fueron encontradas en el océano profundo. Hasta ahora, las actividades mineras (Deep Sea Mining, DSM) no han empezado. Un impacto del DSM podría ser el ruido subacuático. Teniendo en cuenta las frecuencias de ruido de las actividades antrópicas en el océano, y la directiva europea sobre el control del ruido submarino, molestamos los animales con cuatro ruidos acústicos de 1/3 de banda alrededor: 63 Hz, 125 Hz, 500 Hz, 1kHz. Estudiamos los efectos en juveniles de Sparus aurata y en Mytilus galloprovincialis. Analizaremos respuestas comportamiental,moleculares y bioquímicas.

Purpose of this study was the development of a 3D material to be used as substrate for breast cancer cell culture. We developed composite gels constituted by different concentrations of Alginate (A) and Matrigel (M) to obtain a structurally stable-in-time and biologically active substrate. Human aggressive breast cancer cells (i.e. MDA-MB-231) were cultured within the gels. Known the link between cell morphology and malignancy, cells were morphologically characterized and their invasiveness correlated through an innovative bioreactor-based invasion assay. A particular type of gel (i.e. 50% Alginate, 50% Matrigel) emerged thanks to a series of significant results: 1. cells exhibited peculiar cytoskeleton shapes and nuclear fragmentation characteristic of their malignancy; 2. cells expressed the formation of the so-called invadopodia, actin-based protrusion of the plasma membrane through which cells anchor to the extracellular matrix; 3. cells were able to migrate through the gels and attach to an engineered membrane mimicking the vascular walls hosted within bioreactor, providing a completely new 3D in vitro model of the very precursor steps of metastasis.

Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150–4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150–200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.

The effect of euplotin C -- a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus -- on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent (p

The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

Microarray techniques are successfully used to investigate thousands gene expression profiling in a variety of genomic analyses such as gene identification, drug discovery and clinical diagnosis, providing a large amount of genomic data for the overall research community. A Grid based Environment for distributed Microarray data Management and Analysis (GEMMA) is being built. This platform is planned to provide shared, standardized and reliable tools for managing and analyzing biological data related to bone marrow stem cell cultures, in order to maximize the results of distributed experiments. Different microarray analysis algorithms may be offered to the end-user, through a web interface. A set of modular and independent applications may be published on the portal, and either single algorithms or a combination of them might be invoked by the user, through a workflow strategy. Services may be implemented within an existing Grid computing infrastructure to solve problems concerning both large datasets storage (data intensive problem) and large computational times (computing intensive problem). Moreover, experimental data annotation may be collected according to the same rules and stored through the Grid portal, by using a metadata schema, which allows a comprehensive and replicable sharing of microarray experiments among different researchers. The environment has been tested, so far, as regards performance results concerning Grid parallelization of a microarray based gene expression analysis. First results show a very promising speedup ratio.

International audience; This paper reports on an analysis of the bioinformatics and medical informatics literature with the objective to identify upcoming trends that are shared among both research fields to derive benefits from potential collaborative initiatives for their future. Our results present the main characteristics of the two fields and show that these domains are still relatively separated.

The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively.

The presence of opioid, b-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about c-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABAA-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for a1-, a2-, a3-, a6-, b2/3-, c2-, e-, k-, and h-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the a6-, b2/3- and c2-subunits was more intense than the a1-, e-, and h-subunits, suggesting that the former are present in higher concentrations than the latter. � 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The confocal microscope can image a specimen in its natural environment forming a 3D image of the whole structure by scanning it and collecting light through a small aperture (pinhole), allowing in vivo and in vitro observations. So far, the confocal fluorescence microscope (CFM) is considered a true volume imager because of the role of the pinhole that rejects information coming from out-of-focus planes. Unfortunately, intrinsic imaging properties of the optical scheme presently employed yield a corrupted image that can hamper quantitative analysis of successive image planes. By a post-image collection restoration, it is possible to obtain an estimate, with respect to a given optimization criterium, of the true object, utilizing the impulse response of system or Point Spread Function (PSF). The PSF can be measured or predicted so as to have a mathematical and physical model of the image-formation process. Further modelling and recording noise as an additive Gaussian process has used the regularized Iterative Constrained Tykhonov Miller (ICTM) restoration algorithm for solving the inverse problem. This algorithm finds the best estimate iteratively searching among the possible positive solutions; in the Fourier domain, such an approach is relatively fast and elegant. In order to compare the effective improvement in the quantitative image information analysis, we measured the volume of reference objects before and after image restoration, using the isotropic Fakir method.

In order to guarantee an effective and punctual medical intervention to injured people involved in health emergency situations, where usually both professional and non-professional health operators are involved, a fast and accurate treatment has to be carried out. In case of catastrophic or very critical situations, non-professional operators who did not receive proper training (volunteers are among them) could be affected by psychological inhibitions. Their performances could slow down in such way that would affect the quality of the treatment and increase both direct and indirect costs. Our virtual reality system that is currently in use at the health care emergency center of San Martino Hospital in Genoa, Italy, has been designed and developed to check health emergency operators' capabilities to adopt correct decision-making procedures, to make optimal use of new technological equipment and to overcome psychological barriers. Our system is composed of (1) a high-end simulation PC, whose main functions are execution of the main software module, rendering of 3D scenes in stereo mode, rendering of sound, and control of data transmission from/to VR devices; (2) a low-end control PC, which controls the VR simulation running on the simulation PC, manages medical emergency simulation scenarios, introduces unexpected events to the simulation and controls the simulation difficulty level; (3) a magnetic-based motion tracking device used for head and hand tracking; (4) a wireless pair of shutter glasses together with a cathode ray tube wall projector; and (5) a high-end surround sound system. The expected benefits have been verified through the design and implementation of controlled clinical trials.

The fluorochrome 3,3′-dihexyloxacarbocyanine iodide [DiOC 6 (3)], a vital dye utilized to stain the endoplasmic reticulum (ER) of animal and plant cells, has been used to visualize the ER-type structures of Paramecium primaurelia under confocal laser scanning microscopy (CLSM). The morphology of the ER has been studied in paramecia in different physiological conditions. Cells are analysed in early and late logarithmic growth phases, in stationary and in death phases, during shift-up by refeeding after starvation and shift-down by using a starvation medium. In log-phase growing paramecia, the ER constitutes an anastomosing membrane system consisting of short tubules and flattened sacs forming a peripheral network, which is abundant in the cortical region around the trichocysts and the ciliary basal bodies. The tubular network and cytoplasmic membranes are reduced in stationary-phase cells; the original conditions are restored in starved cells after refeeding. The analysis of serial optical sections collected by CLSM at 0.5 μm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells.

BACKGROUND In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities. Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells. The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids. METHODS Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states. RESULTS Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells). These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells). In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells. CONCLUSIONS Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity. A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells. CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.

No quantitative methods are currently available to measure different aggregation parameters in cell cultures. In this paper we describe a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension. The method allows determination, on the same sample at different time intervals, of quantitative parameters, including aggregation percentage, average number of cells in aggregates, and aggregate size statistical distribution. To determine the number of cells in an aggregate starting from its two-dimensional microscopic profile, a model has been proposed and verified, using sphere packing theory. Algorithms have been tested on chondrocyte suspension cultures, where cell aggregation is a very early and critical event leading to cell differentiation. Using this technique for the analysis of chick embryo chondrocyte cultures, we observed that aggregate size and development kinetics depend on the culture conditions used. The method, with minor adaptations, is of potential use also in other cell systems to evaluate aggregation indexes or to study aggregation kinetics.

We have used a confocal laser scanning optical microscope imaging device and a bioimage-oriented workstation equipped for augmented reality to study the helical sperm head of the octopus Eledone cirrhosa. This approach allows us to study different complex organisational motifs due to the spatial arrangement of linear helical structures. We consider this helical specimen an enlarged copy of one of the most important biostructures governing cell functioning such as chromatin-DNA. Moreover, this very same sample is made of highly compacted chromatin that can be studied at higher resolution, i.e., by means of scanning force microscopy. Fluorescence optical sectioning has been used to enter the spatial organisation. Three-dimensional images of single, twisted, and folded fibers are shown.

Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical microscope (CLSM) to display and analyze formation, movement, and fusion of vesicles during the phagocytosis of Paramecium primaurelia, in the x-y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intracellular movement of food vacuoles from their formation at the cytostome to their egestion at the cytoproct was visualized, and food vacuoles were selected in a specific digestion stage. Small pinocytic vesicles are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enlarge the membrane of the nascent food vacuoles or fuse with stage II food vacuoles, when the vacuoles of stage II increase their size, changing from an acidic to an alkaline status. A multimodal analysis of confocal fluorescence images and the false-color technique were used to visualize vesicle movement vs. time. Starting from three images of the same cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color corresponding to each sampling point in time. The composite image shows that vesicles move away from the food vacuole in a scattered manner exhibiting changes in direction.

The Ca 2+ concentration in the cytosol of the marine ciliate protozoan Euplotes crassus has been evaluated utilizing the dye Fluo3-AM and measuring the fluorescent response by confocal laser scanning microscopy (CLSM). Serial sections were piled-up in a three dimensional image of the cell to estimate the cation intracellular spatial distribution and subsequently processed in order to quantify the free Ca 2+ content. The results clearly demonstrated that nano-to micromolar concentrations of Hg 2+ added to seawater culture medium caused up to a 12-fold increase in the free cytosolic Ca 2+ of Euplotes crassus . Exposure of the protozoa to Cu 2+ , an essential element, also caused a sustained increase in the free cytosolic Ca 2+ . Pretreatment of Euplotes crassus with W5, a voltage-dependent Ca 2+ -channel blocker, greatly reduced the deleterious effects of the heavy metals on Ca 2+ concentration. These results indicate that the increase of free Ca 2+ concentration in the cytosol of the protozoa, induced by micromolar heavy metal concentrations, could depend on an increased influx of extracellular Ca 2+ not physiologically compensated by the activity of the different mechanisms involved in Ca 2+ -homeostasis.

An exhaustive description of most biological data requires, besides a set of analytic information, an eidetic representation of the data itself. In this paper, two topics are presented. The first one is confocal microscopy, an advanced technique to produce multidimensional cellular and subcellular structures images. The second one is a software application, named BIOCELL, based on a relational database management system, developed to provide the biologist with an integrated tool to handle simultaneously cell biology linguistic and eidetic information.

Biomedical image analysis workstations can be linked to 3D data-oriented devices for a new approach to image manipulation in biology and medicine. Stereo monitors allow an intuitive approach to medical diagnosis. The use of 3D head-tracking devices allows a more compelling 3D illusion to be generated. A stylus can be used as an electronic knife for dissecting a 3D data set; furthermore, other 3D sensors are available for tracking operator arm movements. The overall character of this work is firmly application oriented, in order to provide concrete operational tools to the medical user. Such tools range from diagnostic up to therapeutic and robotized use of bioimages.

Background Tissue MicroArray technology aims to perform immunohistochemical staining on hundreds of different tissue samples simultaneously. It allows faster analysis, considerably reducing costs incurred in staining. A time consuming phase of the methodology is the selection of tissue areas within paraffin blocks: no utilities have been developed for the identification of areas to be punched from the donor block and assembled in the recipient block. Results The presented work supports, in the specific case of a primary subtype of breast cancer (tubular breast cancer), the semi-automatic discrimination and localization between normal and pathological regions within the tissues. The diagnosis is performed by analysing specific morphological features of the sample such as the absence of a double layer of cells around the lumen and the decay of a regular glands-and-lobules structure. These features are analysed using an algorithm which performs the extraction of morphological parameters from images and compares them to experimentally validated threshold values. Results are satisfactory since in most of the cases the automatic diagnosis matches the response of the pathologists. In particular, on a total of 1296 sub-images showing normal and pathological areas of breast specimens, algorithm accuracy, sensitivity and specificity are respectively 89%, 84% and 94%. Conclusions The proposed work is a first attempt to demonstrate that automation in the Tissue MicroArray field is feasible and it can represent an important tool for scientists to cope with this high-throughput technique.

The authors address using the concept of multimodal bioimage integration at the local area network level, using 3D MRI and PET data of the human head. Here, integration is intended between structural and functional multidimensional images of the same body area of a patient. They analyze the role of present technology and standards (such as the X Window protocol) in providing the physician with a better understanding of the structure-to-function relationships of a given biological organ. They analyze the concept of integration intended as a tool to extract the significative information content from bioimages, i.e. to perform image analysis. The fusion of visual eidetic and linguistic knowledge, which plays an important role in medical diagnosis formation, is seen as a significant step towards obtaining quantitative evaluation of the relevant information content of bioimages. >

Background The SYMBIOmatics Specific Support Action (SSA) is "an information gathering and dissemination activity" that seeks "to identify synergies between the bioinformatics and the medical informatics" domain to improve collaborative progress between both domains (ref. to http://www.symbiomatics.org). As part of the project experts in both research fields will be identified and approached through a survey. To provide input to the survey, the scientific literature was analysed to extract topics relevant to both medical informatics and bioinformatics. Results This paper presents results of a systematic analysis of the scientific literature from medical informatics research and bioinformatics research. In the analysis pairs of words (bigrams) from the leading bioinformatics and medical informatics journals have been used as indication of existing and emerging technologies and topics over the period 2000–2005 ("recent") and 1990–1990 ("past"). We identified emerging topics that were equally important to bioinformatics and medical informatics in recent years such as microarray experiments, ontologies, open source, text mining and support vector machines. Emerging topics that evolved only in bioinformatics were system biology, protein interaction networks and statistical methods for microarray analyses, whereas emerging topics in medical informatics were grid technology and tissue microarrays. Conclusion We conclude that although both fields have their own specific domains of interest, they share common technological developments that tend to be initiated by new developments in biotechnology and computer science.

GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts. © 2005 Wiley Periodicals, inc.

The objective of this study was to develop a software application that is able to support plastic surgeons interested in applying simulations and soft tissue modeling during presurgical planning activities. By using our user-friendly interface and force-displacement graphs, users can analyze scalp skin behaviors when subjected to stress. Users can 1) determine the dynamic of a general point of scalp skin after the application of a specific force, 2) predict the force needed for displacement of a point, and 3) study the deformation of all the points of the entire scalp based on the force applied. Results showed how users are able to manipulate and analyze mechanical behaviors on a mechanical model rather than on a pure geometric qualitative physiological model. The entire application was developed on a Silicon Graphics workstation.

Purpose of this study was the analysis of the role of density and pore interconnection pathway in scaffolds to be used as bone substitutes. We have considered 2 hydroxyapatite bioceramics with identical microstructure and different macro-porosity, pore size distribution and pore interconnection pathway. The scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold A), and (b) foaming (scaffold B). Bone ingrowth within the two bioceramics was obtained using an established model of in vivo bone formation by exogenously added osteoprogenitor cells. The histological analysis of specimens at different time after in vivo implantation revealed in both materials a significant extent of bone matrix deposition. Interestingly enough, scaffold B allowed a faster occurrence of bone tissue, reaching a steady state as soon as 4 weeks. Scaffold A on the other hand reached a comparable level of bone formation only after 8 weeks of in vivo implantation. Both scaffolds were well vascularised, but larger blood vessels were observed in scaffold A. Here we show that porosity and pore interconnection of osteoconductive scaffolds can influence the overall amount of bone deposition, the pattern of blood vessels invasion and finally the kinetics of the bone neoformation process.

Biomedical images play an important role in clinical practice not only at diagnostic but at therapeutic level as well. In this last context, 3D and 4D anatomical and physiological data sets from multimodality imaging techniques need to be integrated and processed. In this work, a standard networking interface protocol such as the X window system has been used as a suitable environment for simultaneous image display. Furthermore, a model for visual and symbolic integration at voxel level is presented. The X window system, even if suffering of several limitations for graphic applications in the multi-D case, offers interesting features in relationship to this perspective, considering the situation of local area network connected workstations. It allows standardization of the user interface and software portability between various hardware nodes, resulting in a friendly and flexible tool for the final medical user.

In this paper we present a theoretical model used in the simulation of the dynamic behaviour of contractile gel fibers. The results of the simulation have been compared with the ones obtained from laboratory experiments. The substantial agreement of these results shows that the model is sufficiently correct to be used in the planning of control strategies for a new class of devices based on polymeric actuators. The developmcnt of such a class of devices is of particular interest for medical applications, owing to the good weight to power ratio, the attainable degree of miniaturization, built in compliance and biocompatible properties of these polymeric materials.

Single energy X-ray imaging, due to its low cost and flexibility, is one of the most used and common technique to assess bone state and bone remodeling over time. Standardized X-ray images are needed to compare sets of radiographs for semi-quantitative analyses of tissue remodeling. However, useful mathematical modeling for the analysis of high level radiographic images are not easily available. In order to propose a useful evaluation tool to a wide clinical scenario, we present an innovative calibration algorithm for a semi-quantitative analysis of non-standardized digitized X-ray images. For calibration on a unique standardization scale, three time invariant regions (ROI) of radiographs were selected and analyzed. The accuracy of the normalization method for X-ray films was successfully validated by using an aluminum step wedge for routine X-ray exposures as tool to standardize serial radiographs (Pearson correlation test: R(2) = 0.96). This method was applied to investigate the progression of the new bone deposition within ceramic scaffolds used as osteoconductive substitute in large bone defects taking advantage of a large animal model. This innovative image-processing algorithm allowed the identification and semi-quantification of the bone matrix deposited within the implant. The osteo-integration at the bone-implant interface was also investigated. A progressively increasing bone tissue deposition within the porous bioceramic implant and a progressive osteo-integration was observed during the 12 months of the trial.

Three-dimensional confocal laser scanning microscopy (CLSM) and two-photon excitation microscopy (TPEM) wereused to study the response of cellular systems to fuzzy organized nanostructured polyelectrolytes used both asmicrocontainers and microcarriers for drug delivery. These nanostructured systems are named Nanocapsules andrepresent a new class of controllable colloids. CLSM and TPEM uniquely allow to follow the fate of encapsulatedliving cells and to track the pathway of nanocapsules introduced into cellular systems.For the former situation, it will be shown how living cells can be encapsulated and demonstrated the preservation of themetabolic and duplicating activity. In this case the role of the Nanocapsule is as microcontainer endowed offunctionalized surface and of protective ability. The latter situation, is related to feeding living cells with Nanocapsules.This experiment serves in elucidating the comprehension of the potential cytotoxicity and of the ability of Nanocapsulesto reach specific targets where active compounds can be released. Cellular systems used within this research areSaccharomyces cerevisiae and Paramecium primaurelia living cells. In the case of encapsulation of Saccharomycescerevisiae living cells, the most relevant result is that, after encapsulation, cells preserve their metabolic activities andthey are still able to divide. At this stage is also relevant the utilization of spectroscopic methods like fluorescencelifetime and second harmonic imaging. These hybrid polyelectrolyte-cells can provide a cheap model system in a widerange of biophysical and biotechnological applications, thanks to the tunable properties of the polyelectrolyte shell.Keywords : Confocal Microscopy; Two-photon excitation microscopy; Nanocapsule; Cell encapsulation; Yeast cells;Protozoa; Endocytosis; Phagocytotsis; Smart Drug Delivery; Fluorescence methods.1. INTRODUCTIONConfocal and Two-photon excitation (1- and 2- photon excitation, respectively) laser scanning microscopy

More than 10 years ago, Tart (1990) described virtual reality (VR) as a technological model of consciousness offering intriguing possibilities for developing diagnostic, inductive, psychotherapeutic, and training techniques that can extend and supplement current ones. To exploit and understand this potential is the overall goal of the "Telemedicine and Portable Virtual Environment in Clinical Psychology"--VEPSY UPDATED--a European Community-funded research project (IST-2000-25323, www.cybertherapy.info). Particularly, its specific goal is the development of different PC-based virtual reality modules to be used in clinical assessment and treatment of social phobia, panic disorders, male sexual disorders, obesity, and eating disorders. The paper describes the clinical and technical rationale behind the clinical applications developed by the project. Moreover, the paper focuses its analysis on the possible role of VR in clinical psychology and how it can be used for therapeutic change.

In Paramecium, internal Ca(2+) concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca(2+) influx, and the addition of drugs that block the Ca(2+) current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABA(B) receptors in P. primaurelia swimming control. The presence of GABA(B) immunoanalogue in Paramecium was evidenced using SDS-PAGE, Western blotting, and confocal laser scanning microscopy. By applying the specific GABA(B) receptor agonist baclofen, a dose-dependent inhibition of the membrane depolarization-induced CR duration was observed. This inhibition was antagonized by phaclofen, persisted when K(+) channel blockers were applied, and disappeared after treatment with nifedipine and verapamil. Moreover, the action of baclofen on depolarization-induced CR was suppressed by treatment with pertussis toxin. Therefore, these experiments suggest that baclofen modulates CR by a G protein (G(0) or G(1)) mediated inhibition of dihydropyridine-sensible calcium channels. Finally, synthesis and release of GABA in the environment by Paramecium have been demonstrated by HPLC. Possible correlations between GABA(B) receptor activation and the regulation of intracellular Ca(2+) levels are discussed.

Summary In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY® FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 µm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.