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.

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.

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.

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.

Time-variant analysis of organelle and vesicle movement during phagocytosis in Paramecium primaurelia by means of fluorescence confocal laser scanning microscopy.

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.

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.

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.

Image analysis of lysosomal activity during the early clonal life of Paramecium primaurelia.

Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.

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.

Acid phosphatase activity in mating type I and mating type II cell lines of Paramecium primaurelia.

The cellular acid phosphatase content, a marker enzyme for lysosomal activity, in Paramecium primaurelia mating type I and mating type II cells was determined by optical laser scanning microscopy. The naphthol AS-TR phosphatase-hexazotized pararosaniline method was used to visualize acid phosphatase activity by the light microscopy. Cell lines of both mating types were tested during culture life, from the early log phase to the death phase. The amount of acid phosphatase was higher in mating type II than in mating type I until the onset of the stationary phase, and then the values reversed. Indeed, during the log phase of growth, mating type II cells formed a higher number of food vacuoles, so that, by taking up a higher amount of bacteria, they sooner became deprived of food. It is suggested that, by lacking nutrients, their synthesis activities and acid phosphatase content were reduced as compared with mating type I cells.

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.

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.

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.