Active bone marrow segmentation based on computed tomography imaging in anal cancer patients: A machine-learning-based proof of concept.

PURPOSE: Different methods are available to identify haematopoietically active bone marrow (ActBM). However, their use can be challenging for radiotherapy routine treatments, since they require specific equipment and dedicated time. A machine learning (ML) approach, based on radiomic features as inputs to three different classifiers, was applied to computed tomography (CT) images to identify haematopoietically active bone marrow in anal cancer patients. METHODS: A total of 40 patients was assigned to the construction set (training set + test set). Fluorine-18-Fluorodeoxyglucose Positron Emission Tomography (18FDG-PET) images were used to detect the active part of the pelvic bone marrow (ActPBM) and stored as ground-truth for three subregions: iliac, lower pelvis and lumbosacral bone marrow (ActIBM, ActLPBM, ActLSBM). Three parameters were used for the correspondence analyses between 18FDG-PET and ML classifiers: DICE index, Precision and Recall. RESULTS: For the 40-patient cohort, median values [min; max] of the Dice index were 0.69 [0.20; 0.84], 0.76 [0.25; 0.89], and 0.36 [0.15; 0.67] for ActIBM, ActLSBM, and ActLPBM, respectively. The Precision/Recall (P/R) ratio median value for the ActLPBM structure was 0.59 [0.20; 1.84] (over segmentation), while for the other two subregions the P/R ratio median has values of 1.249 [0.43; 4.15] for ActIBM and 1.093 [0.24; 1.91] for ActLSBM (under segmentation). CONCLUSION: A satisfactory degree of overlap compared to 18FDG-PET was found for 2 out of the 3 subregions within pelvic bones. Further optimization and generalization of the process is required before clinical implementation.

Long-range phase synchronization of high-frequency oscillations in human cortex.

Inter-areal synchronization of neuronal oscillations at frequencies below ~100 Hz is a pervasive feature of neuronal activity and is thought to regulate communication in neuronal circuits. In contrast, faster activities and oscillations have been considered to be largely local-circuit-level phenomena without large-scale synchronization between brain regions. We show, using human intracerebral recordings, that 100-400 Hz high-frequency oscillations (HFOs) may be synchronized between widely distributed brain regions. HFO synchronization expresses individual frequency peaks and exhibits reliable connectivity patterns that show stable community structuring. HFO synchronization is also characterized by a laminar profile opposite to that of lower frequencies. Importantly, HFO synchronization is both transiently enhanced and suppressed in separate frequency bands during a response-inhibition task. These findings show that HFO synchronization constitutes a functionally significant form of neuronal spike-timing relationships in brain activity and thus a mesoscopic indication of neuronal communication per se.

Pediatric Brain Tissue Segmentation from MRI using Clustering: a Preliminary Study.

Brain Tissue Segmentation (BTS) in young children and neonates is not a trivial task due to peculiar characteristics of the developing brain. The aim of this study is to present the preliminary results of new atlas-free BTS (afBTS) algorithm of MR images for pediatric applications, based on clustering. The algorithm works on axial T1, T2 and FLAIR sequences. First, the Cerebrospinal Fluid (CSF) is identified using the Region Growing algorithm. The remaining voxels are processed with the k-means algorithm in order to separate White Matter (WM) and Grey Matter (GM). The afBTS algorithm was applied to a population of 13 neonates; the segmentations were evaluated by two expert pediatric neuroradiologists and compared with an atlas-based algorithm. The results were promising: afBTS allowed reconstruction of WM and CSF with an image quality comparable to the reference of standard while lower segmentation quality was obtained for the GM segmentation.

Structural Connectivity Analysis in Children with Segmental Callosal Agenesis.

BACKGROUND AND PURPOSE: Segmental callosal agenesis is characterized by the absence of the intermediate callosal portion. We aimed to evaluate the structural connectivity of segmental callosal agenesis by using constrained spherical deconvolution tractography and connectome analysis. MATERIALS AND METHODS: We reviewed the clinical-radiologic features of 8 patients (5 males; mean age, 3.9 years). Spherical deconvolution and probabilistic tractography were performed on diffusion data. Structural connectivity analysis, including summary network metrics, modularity analysis, and network consistency measures, was applied in 5 patients and 10 age-/sex-matched controls. RESULTS: We identified 3 subtypes based on the position of the hippocampal commissure: beneath the anterior callosal remnant in 3 patients (type I), beneath the posterior callosal remnant in 3 patients (type II), and between the anterior and posterior callosal remnants in 2 patients (type III). In all patients, the agenetic segment corresponded to fibers projecting to the parietal lobe, and segmental Probst bundles were found at that level. Ectopic callosal bundles were identified in 3 patients. Topology analysis revealed reduced global connectivity in patients compared with controls. The network topology of segmental callosal agenesis was more variable across patients than that of the control connectomes. Modularity analysis revealed disruption of the structural core organization in the patients. CONCLUSIONS: Three malformative subtypes of segmental callosal agenesis were identified. Even the absence of a small callosal segment may impact global brain connectivity and modularity organization. The presence of ectopic callosal bundles may explain the greater interindividual variation in the connectomes of patients with segmental callosal agenesis.

Gamma-amino butyric acid (GABA) release in the ciliated protozoon Paramecium occurs by neuronal-like exocytosis.

Paramecium primaurelia expresses a significant amount of gamma-amino butyric acid (GABA). Paramecia possess both glutamate decarboxylase (GAD)-like and vesicular GABA transporter (vGAT)-like proteins, indicating the ability to synthesize GABA from glutamate and to transport GABA into vesicles. Using antibodies raised against mammalian GAD and vGAT, bands with an apparent molecular weight of about 67 kDa and 57 kDa were detected. The presence of these bands indicated a similarity between the proteins in Paramecium and in mammals. VAMP, syntaxin and SNAP, putative proteins of the release machinery that form the so-called SNARE complex, are present in Paramecium. Most VAMP, syntaxin and SNAP fluorescence is localized in spots that vary in size and density and are primarily distributed near the plasma membrane. Antibodies raised against mammal VAMP-3, sintaxin-1 or SNAP-25 revealed protein immunoblot bands having molecular weights consistent with those observed in mammals. Moreover, P. primaurelia spontaneously releases GABA into the environment, and this neurotransmitter release significantly increases after membrane depolarization. The depolarization-induced GABA release was strongly reduced not only in the absence of extracellular Ca(2+) but also by pre-incubation with bafilomycin A1 or with botulinum toxin C1 serotype. It can be concluded that GABA occurs in Paramecium, where it is probably stored in vesicles capable of fusion with the cell membrane; accordingly, GABA can be released from Paramecium by stimulus-induced, neuronal-like exocytotic mechanisms.

Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system.

In this study, we investigated the effect of the long-term (10 days) application of a defined and uniform level of fluid flow (uniform shear stress of 1.2 x 10(-3) N/m(2)) on human bone marrow stromal cells (BMSC) cultured on different substrates (i.e., uncoated glass or calcium phosphate coated glass, Osteologictrade mark) in a 2D parallel plate model. Both exposure to flow and culture on Osteologic significantly reduced the number of cell doublings. BMSC cultured under flow were more intensely stained for collagen type I and by von Kossa for mineralized matrix. BMSC exposed to flow displayed an increased osteogenic commitment (i.e., higher mRNA expression of cbfa-1 and osterix), although phenotype changes in response to flow (i.e., mRNA expression of osteopontin, osteocalcin and bone sialoprotein) were dependent on the substrate used. These findings highlight the importance of the combination of physical forces and culture substrate to determine the functional state of differentiating osteoblastic cells. The results obtained using a simple and controlled 2D model system may help to interpret the long-term effects of BMSC culture under perfusion within 3D porous scaffolds, where multiple experimental variables cannot be easily studied independently, and shear stresses cannot be precisely computed.

Improvement in volume estimation from confocal sections after image deconvolution.

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.

JUST in time health emergency interventions: an innovative approach to training the citizen for emergency situations using virtual reality techniques and advanced IT tools (the VR Tool).

This paper reports the results of the second of the two systems developed by JUST, a collaborative project supported by the European Union under the Information Society Technologies (IST) Programme. The most innovative content of the project has been the design and development of a complementary training course for non-professional health emergency operators, which supports the traditional learning phase, and which purports to improve the retention capability of the trainees. This was achieved with the use of advanced information technology techniques, which provide adequate support and can help to overcome the present weaknesses of the existing training mechanisms.

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy.

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 microm 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 degrees 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.

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy.

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.

Three-dimensional computer-aided reconstruction of FMRFamide immunopositive neuron distribution in the ventral ganglion of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

We have implemented a simple program to solve three of the problems related to 3D reconstruction (3D-Rec) of soft tissues: alignment of sections, distortions, and estimation of the spatial position of elements of interest inside the tissues. As a model, we chose the distribution of FMRFamide-like immunopositive neurons in the ventral ganglion of the barnacle Balanus amphitrite collected during different seasonal periods. Images of immunostained sections were acquired by means of a CCD-camera-equipped microscope and a PC and the reference points were taken inside the sections. The FMRFamide-like immunopositive neurons detected in the barnacle ventral ganglion were grouped into four different classes according to size, shape and staining intensity. More numerous FMRFamide-like immunopositive neurons were detected in the autumn-collected barnacle than in the summer counterpart. The two 3D reconstructions obtained from transverse and longitudinal ventral ganglion sections were efficaciously compared after 90 degrees rotation of one of them. Comparison of these two 3D-Rec suggests the presence of at least two groups of FMRFamide-like immunopositive neurons that are seasonally-related and probably involved in reproduction.

Changes in the endoplasmic reticulum structure of Paramecium primaurelia in relation to different cellular physiological states.

The fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC6(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 microm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells.

Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development.

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.

Trichocyst membrane fate in living Paramecium primaurelia visualized by multimodal confocal imaging.

Trichocysts are secretory organelles located at the surface of several ciliates, docked at the plasma membrane. Their secretion is similar to other exocytic processes: the trichocyst membrane fuses with the plasma membrane, its content is released outside the cell, and the membrane is retrieved back into the cell. The fate of the trichocyst membrane in living Paramecium primaurelia was investigated by inducing massive synchronous exocytosis in the presence of fluorescein isothiocyanate-conjugated lectins or of cationized ferritin. The marker is trapped within the retrieved trichocyst membrane sac, and many regularly spaced, fluorescent ghosts are formed. As time proceeds, the number of labeled ghosts decreases, and few fluorescent vacuoles appear within the cell. The relationship between trichocyst ghosts and the vesicles of the phagosome-lysosome system was examined by labeling cells with Texas Red-conjugated bovine serum albumin, a fluorescent marker for phagocytosis. Starting from two confocal images of the same cell labeled with the two fluorescent probes, a new single image was generated by associating each image with a different red or green value. This multimodal analysis showed that trichocyst ghosts fuse with secondary lysosomes or are incorporated into digestive vacuoles. The vacuolar content is degraded and fluorescence is then found in the vesicles of the phagosome-lysosome system, and, at last, in small weakly labeled vesicles located on the cell surface.

Studies on the structure of sperm heads of Eledone cirrhosa by means of CLSM linked to bioimage-oriented devices.

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.

Simulation of soft-tissue tumor excisions: a multimodal interactive approach.

Total 3-D reconstruction of the tumor size, shape, and relations with surrounding structures using CT, MRI, sonography, and angiography images can make simulated radical resection of soft-tissue sarcomas possible, thus sparing normal tissues. With our approach, starting from three MR images for a given patient, a new single image representation of all three parameters is generated by using two different techniques on a workstation in a standard UNIX and X-11 environment. The first one is a transformation linking together the MR parameters and the RGB (red, green, blue) color components. The second one is an unsupervised segmentation method based on a number of neural and fuzzy models. We can dinamically render and update a stereo display using field sequential presentation of left and right eye views on the monitor, with Cristal Eyes LCD shutter eyewear (StereoGraphics Inc., San Rafael, CA) to view it. As 3D locating tool, a 3D locating control system based on low-frequency magnetic fields (Polhemus Fastrak) has been chosen. Simulations of soft-tissues excisions may be performed in this interactive environment with augmented-reality modalities. All this, in our experience, has greatly facilitated the simulation of soft-tissue sarcoma excisions.

An "augmented-reality" aid for plastic and reconstructive surgeons.

Starting from MR and CT images for a given patient, a new single image representation of all parameters has been generated by using false-color techniques in a standard UNIX and X-11 environment. A transformation linking together the MR, CT parameters and the RGB (red, green, blue) color components has been used. Moreover an unsupervised segmentation method based on a number of neural and fuzzy models may directly produce segmented image volumes. Each image of the various sequences has been interactively displayed by using a specifically designed application. The resulting images have been displayed on a stereo monitor allowing the three-dimensional rendering of visual data through LCD shuttered glasses. Moreover, a 3-D control system based on low frequency magnetic fields has been used, while a bandheld Polhemus stylus could be used as an electronic knife for dissecting the 3-D data set and for defining flaps and grafts. Bone or soft-tissue contour can be analyzed, and sections can be removed from the model to allow a view of the underlying structures. Flaps and grafts obtained utilizing the above-reported techniques can be fitted exactly, without repeated removal and recarving. Nuances of depth, tapering, and arc are carved directly into the bone, while chances of asymmetry are markedly diminished. In this way, moreover, anesthetic times are reduced by more efficient utilization of operative time, which usually offsets the increased cost of imaging.