Improvement of speed-accuracy tradeoff during practice of a point-to-point task in children with acquired dystonia

: The tradeoff between speed and accuracy is a well-known constraint for human movement, but previous work has shown that this tradeoff can be modified by practice, and the quantitative relationship between speed and accuracy may be an indicator of skill in some tasks. We have previously shown that children with dystonia are able to adapt their movement strategy in a ballistic throwing game to compensate for increased variability of movement. Here, we test whether children with dystonia can adapt and improve skills learned on a trajectory task. We use a novel task in which children move a spoon with a marble between two targets. Difficulty is modified by changing the depth of the spoon. Our results show that both healthy children and children with acquired dystonia move more slowly with the more difficult spoons, and both groups improve the relationship between speed and spoon difficulty following 1 wk of practice. By tracking the marble position in the spoon, we show that children with dystonia use a larger fraction of the available variability, whereas healthy children adopt a much safer strategy and remain farther from the margins, as well as learning to adapt and have more control over the marble's utilized area by practice. Together, our results show that both healthy children and children with dystonia choose trajectories that compensate for risk and inherent variability, and that the increased variability in dystonia can be modified with continued practice.NEW & NOTEWORTHY This study provides insights into the adaptability of children with dystonia in learning a point-to-point task. We show that these children adjust their strategies to account for increased difficulty in the task. Our findings underscore the potential of task-specific practice in improving motor skills and show higher level of signal-dependent noise can be controlled through repetition and learned strategies, which provides an avenue for the quantitative evaluation of rehabilitation strategies in this challenging group

Spike detection algorithm improvement, spike waveforms projections with PCA and hierarchical classification

Definition of single spikes from multiunit spike trains plays a critical role in neurophysiology and in neuroengineering. Moreover, long period analysis are needed to study synaptic plasticity effects and observe the long and medium term development on which all central nervous system (CNS) learning functions are based. Therefore, the increasing importance of long period recordings makes necessary on-line and real time analysis, memory use optimization and data transmission rate improvement. A threshold-amplitude spikes detection method is chosen and 5 noise level estimate methods were developed. Than APs are bundled to group using principal component analysis and classified (hierarchical classifier). The system has lot of applications like high-throughput pharmacological screening and monitoring effects

537. New Graph-Based Algorithm for Comprehensive Identification and Tracking Retroviral Integration Sites

Vector integration sites (IS) in hematopoietic stem cell (HSC) gene therapy (GT) applications are stable genetic marks, distinctive for each independent cell clone and its progeny. The characterization of IS allows to identify each cell clone and individually track its fate in different tissues or cell lineages and during time, and is required for assessing the safety and efficacy of the treatment. Bioinformatics pipelines for IS detection used in GT identify the sequence reads mapping in the same genomic position of the reference genome as a single IS but discard those ambiguously mapped in multiple genomic regions. The loss of such significant portion of patients' IS may hide potential malignant events thus reducing the reliability of IS studies. We developed a novel tool that is able to accurately identify IS in any genomic region even if composed by repetitive genomic sequences. Our approach exploits an initial genome free analysis of sequencing reads by creating an undirected graph in which nodes are the input sequences and edges represent valid alignments (over a specific identity threshold) between pairs of nodes. Through the analysis and decomposition of the graph, the method identifies indivisible subgraphs of sequences (clusters), each of them corresponding to an IS. Once extracted the consensus sequence of the clusters and aligned on the reference genome, we collect the alignment results and the annotation labels from RepeatMasker. By combining the set of genomic coordinates and the annotation labels, the method retraces the initial sequence graph, statistically validates the clusters through permutation test and produces the final list of IS. We tested the reliability of our tool on 3 IS datasets generated from simulated sequencing reads with incremental rate of nucleotide variations (0%, 0.25% and 0.5%) and real data from a cell line with known IS and we compared out tool to VISPA and UClust, used for GT studies. In the simulated datasets our tool demonstrated precision and recall ranging 0.85-0.97 and 0.88-0.99 respectively, producing the aggregate F-score ranging 0.86-0.98 which resulted higher than VISPA and UClust. In the experimental case of sequences from LAM-PCR products, our tool and VISPA were able to identify all the 6 known ISs for >98% of the reads produced, while UClust identified only 5 out 6 ISs. We then used our tool to reanalyze the sequencing reads of our GT clinical trial for Metachromatic Leukodystrophy (MLD) completing the hidden portion of IS. The overall number of ISs, sequencing reads and estimated actively re-populating HSCs was increased by an average fold ~1.5 with respect the previously published data obtained through VISPA whereas the diversity index of the population did not change and no aberrant clones in repeats occurred. Our tool addresses and solves important open issues in retroviral IS identification and clonal tracking, allowing the generation of a comprehensive repertoire of IS

Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy

Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translatio

A novel mea bioreactor measuring neural network activity continuously over long periods to study synaptic plasticity and pharmacological outcomes

The ability of culturing neurons for a long time on MicroElectrode Array (MEA) devices plays a critical role in understanding some long-term behaviors of a neuronal network, such as the long-term synaptic plasticity. Moreover, pharmacological outcomes usually requires long recordings to evaluate the complete effects in the culture activity. Applications involving MEAs in long-term analysis suffer from some limits imposed by the current experimental setup. The neurons, cultured on the MEA slices, are housed in an incubator; then they are extracted to record network electrical activity. This procedure can damage the cells (i.e. pH variation, sterility problems, medium evaporation) causing a gradual decline in the health of these cultures and, eventually, the cells apoptosis. Therefore experiments must be limited in time. Lots of solutions are in literature: some of them improve the cells survival but they need of an external incubator; others try to realize independent systems, able to autonomously control temperature, humidity and sterility but not gasses. In this work we present the technological development of an experimental system to measure neural network activity with MEAs continuously over long periods in a controlled atmosphere. The bioreactor described aimed to overcome the above-mentioned limits, in order to provide a single tool that record and process on-line neuronal action potentials without the need of an external incubator. The incubating chamber prototype was designed with Pro-Engineer Wildfire. It was produced, from a cylinder block in polymethylmethacrylate (PMMA - Plexiglas), using rapid prototyping method (Roland Modela MDX-40). The MEA housing and a symmetrical small chamber, the temperature reference, were realised in the bigger one. The heating was obtained bathing a termoresistance in a water bath surrounding the incubating chamber. The gasses and the humidity control (95% Air, 5% CO2; 95% steam) were developed using a commercial CO2 and bubbling module. The electronic for the activity recording was placed on the external top of the device. The whole system was sterilized with Ethylene Oxide (ETO) in order to assure sterility requirement. Software simulations were used to optimize the on-line spike detection and clustering for future implementation on hardware.Preliminary prototype validation on biological environment shows a good capability of cells growth and preservation for long time; moreover, MEA’s electrodes seem not to be spoiled by the incubating environment

Quantitative Acylation of Amino Compounds Catalysed by Penicillin G Acylase in Organic Solvent at Controlled Water Activity

Covalently immobilised penicillin G acylase (PGA-450) accepts in toluene, at controlled water activity (aw), a broad range of amino compounds as nucleophiles in kinetically controlled acylation. Hydrolytic reactions were prevented and complete conversions were achieved in short times even when working with an equimolar concentration of the substrates. The recovery of the products was facile, leading to high isolation yields. The obtained N-acylated derivatives of L-amino acids can be used in further reactions, since no purification steps are required in such conditions. This opens new perspectives to the application of PGA in selective protection of the amino function for peptide synthesis. Ali attempts to perform esterification and transesterification reactions with PGA in toluene, at the same aw as used for the acylation of amino compounds, were unsuccesful

A novel mea bioreactor measuring neural network activity continuously over long periods to study synaptic plasticity and pharmacological outcomes

The ability of culturing neurons for a long time on MicroElectrode Array (MEA) devices plays a critical role in understanding some long-term behaviors of a neuronal network, such as the long-term synaptic plasticity. Moreover, pharmacological outcomes usually requires long recordings to evaluate the complete effects in the culture activity. Applications involving MEAs in long-term analysis suffer from some limits imposed by the current experimental setup. The neurons, cultured on the MEA slices, are housed in an incubator; then they are extracted to record network electrical activity. This procedure can damage the cells (i.e. pH variation, sterility problems, medium evaporation) causing a gradual decline in the health of these cultures and, eventually, the cells apoptosis. Therefore experiments must be limited in time. Lots of solutions are in literature: some of them improve the cells survival but they need of an external incubator; others try to realize independent systems, able to autonomously control temperature, humidity and sterility but not gasses. In this work we present the technological development of an experimental system to measure neural network activity with MEAs continuously over long periods in a controlled atmosphere. The bioreactor described aimed to overcome the above-mentioned limits, in order to provide a single tool that record and process on-line neuronal action potentials without the need of an external incubator. The incubating chamber prototype was designed with Pro-Engineer Wildfire. It was produced, from a cylinder block in polymethylmethacrylate (PMMA - Plexiglas), using rapid prototyping method (Roland Modela MDX-40). The MEA housing and a symmetrical small chamber, the temperature reference, were realised in the bigger one. The heating was obtained bathing a termoresistance in a water bath surrounding the incubating chamber. The gasses and the humidity control (95% Air, 5% CO2; 95% steam) were developed using a commercial CO2 and bubbling module. The electronic for the activity recording was placed on the external top of the device. The whole system was sterilized with Ethylene Oxide (ETO) in order to assure sterility requirement. Software simulations were used to optimize the on-line spike detection and clustering for future implementation on hardware.Preliminary prototype validation on biological environment shows a good capability of cells growth and preservation for long time; moreover, MEA’s electrodes seem not to be spoiled by the incubating environment

442. LV Expressing MR Reporter Genes Allows In Vivo Monitoring of Stem Cell Gene Therapy

Somatic stem cells (SSC) have raised interest because of their therapeutic potential in both cell-based and gene therapy applications. Towards this goal, tracking the fate of either delivered cells or of genetically modified endogenous cells is of utmost importance. Diverse imaging approaches are available for cell tracking and among these MRI shows a greater resolution and allows direct anatomic correlation and long-term studies of dynamic cell migration on living animals. Superparamagnetic iron oxide (SPIO) has been used to label SSC in vitro and to make them detectable in vivo upon transplantation. However, major limitations of this approach are the progressive dilution of the contrast media among cell progeny and the need for ex vivo SPIO loading. We thus explored an alternative strategy based on the combination of lentiviral vectors (LV), which efficiently transduce SSC both ex vivo and in vivo and allow long-term expression in their progeny, and MR reporter genes, able to increase iron uptake and accumulation into different cell types

A framework for the comparative assessment of neuronal spike sorting algorithms towards more accurate off-line and on-line microelectrode arrays data analysis

Neuronal spike sorting algorithms are designed to retrieve neuronal network activity on a single-cell level from extracellular multiunit recordings with Microelectrode Arrays (MEAs). In typical analysis of MEA data, one spike sorting algorithm is applied indiscriminately to all electrode signals. However, this approach neglects the dependency of algorithms' performances on the neuronal signals properties at each channel, which require data-centric methods. Moreover, sorting is commonly performed off-line, which is time and memory consuming and prevents researchers from having an immediate glance at ongoing experiments. The aim of this work is to provide a versatile framework to support the evaluation and comparison of different spike classification algorithms suitable for both off-line and on-line analysis. We incorporated different spike sorting "building blocks" into a Matlab-based software, including 4 feature extraction methods, 3 feature clustering methods, and 1 template matching classifier. The framework was validated by applying different algorithms on simulated and real signals from neuronal cultures coupled to MEAs. Moreover, the system has been proven effective in running on-line analysis on a standard desktop computer, after the selection of the most suitable sorting methods. This work provides a useful and versatile instrument for a supported comparison of different options for spike sorting towards more accurate off-line and on-line MEA data analysis

Relationship between hemoglobin, hemolysis, and transcranial Doppler velocities in children with sickle cell disease: Results from a long-term natural history study in Italy in the era of multimodal therapy

Background: Stroke and cerebral vasculopathy are leading causes of morbidity and mortality in patients with sickle cell disease (SCD). Transcranial Doppler (TCD) is a reliable and validated predictor of stroke risk. Children with conditional or abnormal TCD are at an increased risk for stroke, which can be mitigated by red blood cell transfusion or hydroxyurea. Elucidating the relationship between cerebral hemodynamics and hemolytic anemia can help identify novel therapeutic approaches to reduce stroke risk and transfusion dependence.Methods: This long-term, real-world study was designed to evaluate the prevalence of TCD imaging (TCDi)-assessed flow velocities in children and to interrogate their relationship with markers of anemia and hemolysis.Results: In total, 155 children (median follow-up 79.8 months, 1358.44 patient-years) had 583 evaluable TCDi results. Only patients with HbSS or HbS beta(0) had abnormal (1.6%) or conditional (10.9%) TCDi. Children with abnormal or conditional TCDi had lower hemoglobin (Hb) and higher hemolysis markers. A linear correlation was detected between TCD velocity and Hb: an Hb increase of 1 g/dL corresponded to decreases in velocity in the internal carotid and middle cerebral arteries (6.137 cm/s and 7.243 cm/s). Moreover, patients with Hb >9 g/dL presented a lower risk of TCDi-associated events.Conclusion: These results support the need to optimize disease-modifying treatments that increase Hb and reduce hemolysis for stroke prevention in young children with SCD