Marco Fato

A model to prioritize access to elective surgery on the basis of clinical urgency and waiting time

BackgroundPrioritization of waiting lists for elective surgery represents a major issue in public systems in view of the fact that patients often suffer from consequences of long waiting times. In addition, administrative and standardized data on waiting lists are generally lacking in Italy, where no detailed national reports are available. This is true although since 2002 the National Government has defined implicit Urgency-Related Groups (URGs) associated with Maximum Time Before Treatment (MTBT), similar to the Australian classification. The aim of this paper is to propose a model to manage waiting lists and prioritize admissions to elective surgery.MethodsIn 2001, the Italian Ministry of Health funded the Surgical Waiting List Info System (SWALIS) project, with the aim of experimenting solutions for managing elective surgery waiting lists. The project was split into two phases. In the first project phase, ten surgical units in the largest hospital of the Liguria Region were involved in the design of a pre-admission process model. The model was embedded in a Web based software, adopting Italian URGs with minor modifications. The SWALIS pre-admission process was based on the following steps: 1) urgency assessment into URGs; 2) correspondent assignment of a pre-set MTBT; 3) real time prioritization of every referral on the list, according to urgency and waiting time. In the second project phase a prospective descriptive study was performed, when a single general surgery unit was selected as the deployment and test bed, managing all registrations from March 2004 to March 2007 (1809 ordinary and 597 day cases). From August 2005, once the SWALIS model had been modified, waiting lists were monitored and analyzed, measuring the impact of the model by a set of performance indexes (average waiting time, length of the waiting list) and Appropriate Performance Index (API).ResultsThe SWALIS pre-admission model was used for all registrations in the test period, fully covering the case mix of the patients referred to surgery. The software produced real time data and advanced parameters, providing patients and users useful tools to manage waiting lists and to schedule hospital admissions with ease and efficiency. The model protected patients from horizontal and vertical inequities, while positive changes in API were observed in the latest period, meaning that more patients were treated within their MTBT.ConclusionThe SWALIS model achieves the purpose of providing useful data to monitor waiting lists appropriately. It allows homogeneous and standardized prioritization, enhancing transparency, efficiency and equity. Due to its applicability, it might represent a pragmatic approach towards surgical waiting lists, useful in both clinical practice and strategic resource management.

Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model

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.

Automatic segmentation of deep intracerebral electrodes in computed tomography scans

BackgroundInvasive monitoring of brain activity by means of intracerebral electrodes is widely practiced to improve pre-surgical seizure onset zone localization in patients with medically refractory seizures. Stereo-Electroencephalography (SEEG) is mainly used to localize the epileptogenic zone and a precise knowledge of the location of the electrodes is expected to facilitate the recordings interpretation and the planning of resective surgery. However, the localization of intracerebral electrodes on post-implant acquisitions is usually time-consuming (i.e., manual segmentation), it requires advanced 3D visualization tools, and it needs the supervision of trained medical doctors in order to minimize the errors. In this paper we propose an automated segmentation algorithm specifically designed to segment SEEG contacts from a thresholded post-implant Cone-Beam CT volume (0.4 mm, 0.4 mm, 0.8 mm). The algorithm relies on the planned position of target and entry points for each electrode as a first estimation of electrode axis. We implemented the proposed algorithm into DEETO, an open source C++ prototype based on ITK library.ResultsWe tested our implementation on a cohort of 28 subjects in total. The experimental analysis, carried out over a subset of 12 subjects (35 multilead electrodes; 200 contacts) manually segmented by experts, show that the algorithm: (i) is faster than manual segmentation (i.e., less than 1s/subject versus a few hours) (ii) is reliable, with an error of 0.5 mm ± 0.06 mm, and (iii) it accurately maps SEEG implants to their anatomical regions improving the interpretability of electrophysiological traces for both clinical and research studies. Moreover, using the 28-subject cohort we show here that the algorithm is also robust (error < 0.005 mm) against deep-brain displacements (< 12 mm) of the implanted electrode shaft from those planned before surgery.ConclusionsOur method represents, to the best of our knowledge, the first automatic algorithm for the segmentation of SEEG electrodes. The method can be used to accurately identify the neuroanatomical loci of SEEG electrode contacts by a non-expert in a fast and reliable manner.

Endocytosis of GABAB receptors modulates membrane excitability in the single-celled organism Paramecium.

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.

A Grid-based solution for management and analysis of microarrays in distributed experiments

Several systems have been presented in the last years in order to manage the complexity of large microarray experiments. Although good results have been achieved, most systems tend to lack in one or more fields. A Grid based approach may provide a shared, standardized and reliable solution for storage and analysis of biological data, in order to maximize the results of experimental efforts. A Grid framework has been therefore adopted due to the necessity of remotely accessing large amounts of distributed data as well as to scale computational performances for terabyte datasets. Two different biological studies have been planned in order to highlight the benefits that can emerge from our Grid based platform. The described environment relies on storage services and computational services provided by the gLite Grid middleware. The Grid environment is also able to exploit the added value of metadata in order to let users better classify and search experiments. A state-of-art Grid portal has been implemented in order to hide the complexity of framework from end users and to make them able to easily access available services and data. The functional architecture of the portal is described. As a first test of the system performances, a gene expression analysis has been performed on a dataset of Affymetrix GeneChip® Rat Expression Array RAE230A, from the ArrayExpress database. The sequence of analysis includes three steps: (i) group opening and image set uploading, (ii) normalization, and (iii) model based gene expression (based on PM/MM difference model). Two different Linux versions (sequential and parallel) of the dChip software have been developed to implement the analysis and have been tested on a cluster. From results, it emerges that the parallelization of the analysis process and the execution of parallel jobs on distributed computational resources actually improve the performances. Moreover, the Grid environment have been tested both against the possibility of uploading and accessing distributed datasets through the Grid middleware and against its ability in managing the execution of jobs on distributed computational resources. Results from the Grid test will be discussed in a further paper.

Survival Online: a web-based service for the analysis of correlations between gene expression and clinical and follow-up data

BackgroundComplex microarray gene expression datasets can be used for many independent analyses and are particularly interesting for the validation of potential biomarkers and multi-gene classifiers. This article presents a novel method to perform correlations between microarray gene expression data and clinico-pathological data through a combination of available and newly developed processing tools.ResultsWe developed Survival Online (available at http://ada.dist.unige.it:8080/enginframe/bioinf/bioinf.xml), a Web-based system that allows for the analysis of Affymetrix GeneChip microarrays by using a parallel version of dChip. The user is first enabled to select pre-loaded datasets or single samples thereof, as well as single genes or lists of genes. Expression values of selected genes are then correlated with sample annotation data by uni- or multi-variate Cox regression and survival analyses. The system was tested using publicly available breast cancer datasets and GO (Gene Ontology) derived gene lists or single genes for survival analyses.ConclusionThe system can be used by bio-medical researchers without specific computation skills to validate potential biomarkers or multi-gene classifiers. The design of the service, the parallelization of pre-processing tasks and the implementation on an HPC (High Performance Computing) environment make this system a useful tool for validation on several independent datasets.

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 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.

Consistency of EEG source localization and connectivity estimates

ABSTRACT As the EEG inverse problem does not have a unique solution, the sources reconstructed from EEG and their connectivity properties depend on forward and inverse modeling parameters such as the choice of an anatomical template and electrical model, prior assumptions on the sources, and further implementational details. In order to use source connectivity analysis as a reliable research tool, there is a need for stability across a wider range of standard estimation routines. Using resting state EEG recordings of N=65 participants acquired within two studies, we present the first comprehensive assessment of the consistency of EEG source localization and functional/effective connectivity metrics across two anatomical templates (ICBM152 and Colin27), three electrical models (BEM, FEM and spherical harmonics expansions), three inverse methods (WMNE, eLORETA and LCMV), and three software implementations (Brainstorm, Fieldtrip and our own toolbox). Source localizations were found to be more stable across reconstruction pipelines than subsequent estimations of functional connectivity, while effective connectivity estimates where the least consistent. All results were relatively unaffected by the choice of the electrical head model, while the choice of the inverse method and source imaging package induced a considerable variability. In particular, a relatively strong difference was found between LCMV beamformer solutions on one hand and eLORETA/WMNE distributed inverse solutions on the other hand. We also observed a gradual decrease of consistency when results are compared between studies, within individual participants, and between individual participants. In order to provide reliable findings in the face of the observed variability, additional simulations involving interacting brain sources are required. Meanwhile, we encourage verification of the obtained results using more than one source imaging procedure. HighlightsEEG source imaging results depends on forward and inverse modeling parameters.We quantify the consistency of localization and connectivity metrics across pipelines.Considerable variability in connectivity is seen across inverse methods and toolboxes.Beamformer solutions induce different connectivity patterns than linear inverses.Future studies may employ multiple imaging pipelines to demonstrate reliability.

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. Microsc. Res. Tech. 36:159–164, 1997.

Metabotropic γ-aminobutyric acid (GABAB) receptors modulate feeding behavior in the calcisponge Leucandra aspera

Here, we report the presence of the γ-aminobutyric acid (GABA)-ergic system in the calcisponge Leucandra aspera and examine the cellular localization of the components of this system, including GABA-like receptors using immunofluorescence and confocal microscopy. Furthermore, we demonstrate for the first time that GABA plays a functional role as a messenger in regulating sponge-feeding behavior. We found that both GABA(B) R1 and R2 subunits are present in the choanocytes of sponges as well as in the eso- and endopinacocytes. The functional role of GABA in the feeding behavior of this sponge was tested. The involvement of GABA receptors in the endocytic processes in L. aspera was demonstrated with dextran conjugated to Texas Red as a marker for material ingestion and by treating isolated sponge cells with a GABA(B) receptor agonist and an antagonist. The amount of dextran that was ingested increased in dissociated sponge cells when the GABA(B) receptor agonist baclofen was used, and this stimulatory effect was prevented by treatment with the GABA(B) receptor antagonist phaclofen. The baclofen effect on uptake was blocked by treatment with pertussis toxin, thus indicating a role for G proteins in modulating feeding behavior in L. aspera. Moreover, we found evidence that GABA receptors are involved in the consumption of dissolved organic matter by sponge cells. These findings suggest that GABA receptors and their functional role are highly conservative traits in the animal kingdom prenervous system evolution.