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Spinal muscular atrophy is a motor neuron disorder caused by mutations in SMN1. The reasons for the selective vulnerability of motor neurons linked to SMN (encoded by SMN1) reduction remain unclear. Therefore, we performed deep RNA sequencing on human spinal muscular atrophy motor neurons to detect specific altered gene splicing/expression and to identify the presence of a common sequence motif in these genes. Many deregulated genes, such as the neurexin and synaptotagmin families, are implicated in critical motor neuron functions. Motif-enrichment analyses of differentially expressed/spliced genes, including neurexin2 (NRXN2), revealed a common motif, motif 7, which is a target of SYNCRIP. Interestingly, SYNCRIP interacts only with full-length SMN, binding and modulating several motor neuron transcripts, including SMN itself. SYNCRIP overexpression rescued spinal muscular atrophy motor neurons, due to the subsequent increase in SMN and their downstream target NRXN2 through a positive loop mechanism and ameliorated SMN-loss-related pathological phenotypes in Caenorhabditis elegans and mouse models. SMN/SYNCRIP complex through motif 7 may account for selective motor neuron degeneration and represent a potential therapeutic target.

Aims: 1. to investigate diagnostic and prognostic procedures routinely used by international professionals to assess children with disorders of consciousness (DoC); 2. to explore use and availability of internal and national guidelines for pediatric DoC; 3. to identify international differences in diagnostic/prognostic protocols., Methods: The International Brain Injury Association DoC Special Interest Group emailed a survey link to 43,469 professionals. The survey included questions on diagnostic/prognostic procedures and guidelines for children with DoC., Results: Data on 82 respondents [(50% physicians) primarily from Europe (43.9%)and North America (37.8%)] were analyzed. Common diagnostic tools included the Glasgow Coma Scale for clinical assessment (94%), the Coma Recovery Scale-Revised for outcome measurement (57%), and cerebral MRI (94%). Clinical features used most frequently to inform prognosis varied with patient age. Few respondents used national (28%) admission protocols for children with DoC, and most were unaware of published national guidelines for diagnostic (72%) and prognostic (85%) procedures. Compared to North American respondents, more European respondents were physicians and used neurophysiological data for prognosis., Conclusions: This international survey provides useful information about diagnostic and prognostic procedures currently used for children with DoC and highlights the need for guidelines to promote best practices for diagnosis/prognosis in pediatric DoC.

Purpose: The description of motor changes and clinical evolution of a pediatric cohort with acquired brain injury (ABI) over 5 years by the functional independence measure for children (WeeFIM)., Methods: We analyzed retrospective data from 496 patients (aged 0-18 years) with severe ABI admitted for rehabilitation. WeeFIM scores and disability rating scale (reference scale) were collected at admission, discharge and yearly, up to year 5., Results: Functional limitations gradually reduced after ABI, but children still had residual disabilities at the endpoint. Seventeen percent of patients demonstrated no improvement at WeeFIM. Gait analysis showed that decreased WeeFIM scores in the mobility domain are associated with slower and shorter steps, and increased step width, compensating for imbalance., Conclusions: Five years after ABI, improvement is still found in the mobility domain of WeeFIM. This work adds evidence that margin still exists at least until year 5 after injury for profitably continuing the rehabilitation effort.

Robotically-driven orthoses (RDO) are promising for treating gait impairment in children with hemiplegia after acquired brain injury (ABI). Despite this, existing literature on the employment of RDO in ABI is scanty, and cohorts' age spans throughout the adult age, with no specific focus on the developmental age. We aim to compare a treatment solely based on conventional physiotherapy (CP) with a program combining RDO training with CP, and to examine the effect of time following ABI on recovery. A prospective two-cohorts study was conducted in a rehabilitation hospital. Post-acute and chronic children with hemiplegia due to ABI underwent either: (i) 20 sessions of RDO plus 20 sessions of CP (n = 29), or (ii) 40 of CP (n = 12). Gross Motor Function Measures (GMFM), Functional Assessment Questionnaire (FAQ), 6 Minutes Walk Test and gait analysis (GA) parameters were recorded before and after training. Over all the patients in RDO + CP group, all GMFM domains and FAQ improved after RDO training (p < 0.05). The unaffected limb showed significantly decreased stance, increased step length and reduced anteroposterior center of pressure oscillation; the affected side increased the stride length. ROM hip and knee flex-extension increased bilaterally (p < 0.05 for all). RDO training during the acute/subacute post-injury phase increased motor functional abilities, cadence and velocity of gait (p < 0.05). We conclude that RDO imposes a proximal-to-distal differential effect on the lower limbs, with the hip joint being the most stimulated. RDO training fostered recovery, increasing the quality of gait on the unaffected side. Planning RDO early in the rehabilitation course of pediatric ABI is advantageous. RDO + CP may extend rehabilitation efficacy to the proximal segment of leg and to gait velocity., (Copyright © 2018 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Abstract: Disorders of consciousness may follow brain injury, due to impairments of wakefulness and/or awareness. Polysomnography can identify elements that may be ascribed to impairments of specific neuroanatomical areas. Recognizing which impairments affect each patient is crucial for diagnosis, prognosis, and to select an appropriate therapy. We present a pediatric case of insufficient wakefulness in a patient with severe disability following a pilocytic astrocytoma. Polysomnography was crucial for diagnosis, as it detected a well-structured pattern with daytime sleep initiations in the REM sleep phase. Treatment with modafinil was successful, as confirmed by polysomnography, leading to partial recovery of the patient's consciousness and communication ability. We suggest that polysomnography is a useful diagnostic tool to direct the pharmacotherapy and rehabilitation of states of reduced consciousness., (© 2017 American Academy of Sleep Medicine)

Unexplained blood cytopenias, in particular anemia, are often found in older persons. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes is currently poorly defined. We studied a prospective cohort of patients with unexplained cytopenia with the aim to estimate the predictive value of somatic mutations for identifying subjects with, or at risk of, developing a myeloid neoplasm. The study included a learning cohort of 683 consecutive patients investigated for unexplained cytopenia, and a validation cohort of 190 patients referred for suspected myeloid neoplasm. Using granulocyte DNA, we looked for somatic mutations in 40 genes that are recurrently mutated in myeloid malignancies. Overall, 435/683 patients carried a somatic mutation in at least 1 of these genes. Carrying a somatic mutation with a variant allele frequency ≥0.10, or carrying 2 or more mutations, had a positive predictive value for diagnosis of myeloid neoplasm equal to 0.86 and 0.88, respectively. Spliceosome gene mutations and comutation patterns involving TET2 , DNMT3A , or ASXL1 had positive predictive values for myeloid neoplasm ranging from 0.86 to 1.0. Within subjects with inconclusive diagnostic findings, carrying 1 or more somatic mutations was associated with a high probability of developing a myeloid neoplasm during follow-up (hazard ratio = 13.9, P < .001). The predictive values of mutation analysis were confirmed in the independent validation cohort. The findings of this study indicate that mutation analysis on peripheral blood granulocytes may significantly improve the current diagnostic approach to unexplained cytopenia and more generally the diagnostic accuracy of myeloid neoplasms., (© 2017 by The American Society of Hematology.)

Aim: In this observational study using 3T magnetic resonance imaging (MRI) and diffusion tensor, we investigated the differential effects of pathology, stage of disease, state of consciousness, and aetiology on the modifications of supra- and infra-tentorial white matter tracts and their correlations with clinical scales in paediatric patients with severe non-traumatic brain injury., Method: Diffusion tensor magnetic resonance imaging (DT-MRI) was obtained from seven children with unresponsive wakefulness syndrome (UWS; five males, two females; age at event 5y; standard deviation [SD] 2y 1mo), six children in a minimally conscious state (MCS; three males, three females; age at event 5y 10mo; SD 5y), and 10 healthy children as controls(two males, eight females; age at study 10y 10mo; SD 2y 10mo). Fractional anisotropy, mean, axial, and radial diffusivities were calculated for the corpus callosum, inferior, middle (MCP), and superior cerebellar peduncles (SCP)., Results: DT-MRI parameters from corpus callosum and SCP differed between patients and controls. MCP abnormalities were detected in patients presenting non-traumatic composite aetiology (n=4) versus those suffering from pure anoxia (n=9). The supra-tentorial compartment was more damaged (i.e. decreased fractional anisotropy and increased diffusivities) than the infra-tentorial one. Correlations were found between DT-MRI abnormalities and Glasgow Outcome Scale scores., Interpretation: In paediatric UWS/MCS, the severity of clinical disability correlates with white matter tract abnormalities., (© 2016 Mac Keith Press.)

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with variable clinical course. To predict the clinical outcome, we previously developed a CMML-specific prognostic scoring system (CPSS) based on clinical parameters and cytogenetics. In this work, we tested the hypothesis that accounting for gene mutations would further improve risk stratification of CMML patients. We therefore sequenced 38 genes to explore the role of somatic mutations in disease phenotype and clinical outcome. Overall, 199 of 214 (93%) CMML patients carried at least 1 somatic mutation. Stepwise linear regression models showed that these mutations accounted for 15% to 24% of variability of clinical phenotype. Based on multivariable Cox regression analyses, cytogenetic abnormalities and mutations in RUNX1, NRAS, SETBP1, and ASXL1 were independently associated with overall survival (OS). Using these parameters, we defined a genetic score that identified 4 categories with significantly different OS and cumulative incidence of leukemic evolution. In multivariable analyses, genetic score, red blood cell transfusion dependency, white blood cell count, and marrow blasts retained independent prognostic value. These parameters were included into a clinical/molecular CPSS (CPSS-Mol) model that identified 4 risk groups with markedly different median OS (from >144 to 18 months, hazard ratio [HR] = 2.69) and cumulative incidence of leukemic evolution (from 0% to 48% at 4 years, HR = 3.84) (P < .001). The CPSS-Mol fully retained its ability to risk stratify in an independent validation cohort of 260 CMML patients. In conclusion, integrating conventional parameters and gene mutations significantly improves risk stratification of CMML patients, providing a robust basis for clinical decision-making and a reliable tool for clinical trials., (© 2016 by The American Society of Hematology.)

Study Objective: Sleep patterns of pediatric patients in unresponsive wakefulness syndrome (UWS) have been poorly investigated, and the prognostic potential of polysomnography (PSG) in these subjects is still uncertain. The goal of the study was to identify quantitative PSG indices to be applied as possible prognostic markers in pediatric UWS., Methods: We performed PSG in 27 children and adolescents with UWS due to acquired brain damage in the subacute phase. Patients underwent neurological examination and clinical assessment with standardized scales. Outcome was assessed after 36 mo. PSG tracks were scored for sleep stages and digitally filtered. The spectral difference between sleep and wake was computed, as the percent difference at specific spectral frequencies. We computed (1) the ratio between percent power in the delta and alpha frequency bands, (2) the ratio between alpha and theta frequency bands, and (3) the power ratio index, during wake and sleep, as proposed in previous literature. The predictive role of several clinical and PSG measures was tested by logistic regression., Results: Correlation was found between the differential measures of electroencephalographic activity during sleep and wake in several frequency bands and the clinical scales (Glasgow Outcome Score, Level of Cognitive Functioning Assessment Scale, and Disability Rating Scale) at follow-up; the Sleep Patterns for Pediatric Unresponsive Wakefulness Syndrome (SPPUWS) scores correlated with the differential measures, and allowed outcome prediction with 96.3% of accuracy., Conclusions: The differential measure of electroencephalographic activity during sleep and wake in the beta band and, more incisively, SPPUWS can help in determining the capability to recover from pediatric UWS well before the confirmation provided by suitable clinical scales., (© 2016 American Academy of Sleep Medicine.)

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

Objectives: We aimed (i) to search for qualitative sleep patterns for pediatric unresponsive wakefulness syndrome (SPPUWS) in prolonged polysomnographic (PSG) recordings in children and adolescents with subacute severe disorders of consciousness due to an acquired brain damage; (ii) to investigate the clinical relevance of SPPUWS and of possible neurophysiological markers (rapid eye movement sleep and sleep spindles) in PSG recordings of pediatric patients with unresponsive wakefulness syndrome (UWS)., Methods: We performed a PSG study in 27 children with UWS due to acquired brain damage in the subacute phase. Patients received a full neurological examination and a clinical assessment with standardized scales. In addition, outcome was assessed after 36 months., Results: We identified 6 PSG patterns (SPPUWS) corresponding to increasing neuroelectrical complexity. The presence of an organized sleep pattern, as well as rapid eye movement sleep and sleep spindles, in the subacute stage appeared highly predictive of a more favorable outcome. Correlation was found between SPPUWS and recovery, as assessed by several clinical and rehabilitation scales., Conclusions: Polysomnography can be used as a prognostic tool, as it can help determine the capability to recover from a pediatric UWS and predict outcome well before the confirmation provided by suitable clinical scales.

To explore the presence of differential cortical hemodynamic activations related to cognitive components of actions, we performed a fNIRS (functional Near-Infrared Spectroscopy) study during Observation (O), Execution (E) and Imagination (I) of complex and meaningful (transitive and intransitive) gestures in ecological setting. A pilot sample of 5 healthy adults underwent an event-related study consisting of these 3 different conditions, with O set as first and followed by a randomized presentation of E or I. fNIRS measurements were performed using a 24 channel array of optodes (8 light injectors and 8 detectors) placed over the contralateral central, centro-parietal, parietal and temporal areas. Results showed that the premotor (PMC) and the sensory-motor cortices (SM1) were recruited selectively during E, with levels of oxygenated hemoglobin (oxy-Hb) higher than the other conditions, while the posterior parietal cortex (PPC) showed increased oxy-Hb levels for both E and O. These data suggest that variations in hemodynamic responses can be attributed to different neural processes underpinning these tasks, with PMC and SM1 being more involved in action preparation and performance, and PPC prevalently dedicated to attentive processes related to the execution and observation of limb movements.

To evaluate the feasibility of a fully objective rehabilitative and assessment process of the gait abilities in children suffering from Acquired Hemiplegia (AH), we studied the combined employment of robotic-aided gait training (RAGT) and 3D-Gait Analysis (GA). A group of 12 patients with AH underwent 20 sessions of RAGT in addition to traditional manual physical therapy (PT). All the patients were evaluated before and after the training by using the Gross Motor Function Measures (GMFM), the Functional Assessment Questionnaire (FAQ), and the 6 Minutes Walk Test. They also received GA before and after RAGT+PT. Finally, results were compared with those obtained from a control group of 3 AH children who underwent PT only. After the training, the GMFM and FAQ showed significant improvement in patients receiving RAGT+PT. GA highlighted significant improvement in stance symmetry and step length of the affected limb. Moreover, pelvic tilt increased, and hip kinematics on the sagittal plane revealed statistically significant increase in the range of motion during the hip flex-extension. Our data suggest that the combined program RAGT+PT induces improvements in functional activities and gait pattern in children with AH, and it demonstrates that the combined employment of RAGT and 3D-GA ensures a fully objective rehabilitative program.

We studied 86 patients with severe acquired brain injuries of different etiology, aged 0 to 18 years, in a condition of vegetative state or minimally conscious state. During neurorehabilitation, we administered the Level of Cognitive Functioning Assessment Scale every 2 weeks in order to describe and compare the progressive improvement in their cognitive-behavioral functioning and responsiveness in different etiologies. Patients with traumatic brain injury showed more favorable clinical outcomes. The higher the level of functioning at the first evaluation, the better the outcome, and the higher the Glasgow Coma Scale score, the higher the Level of Cognitive Functioning Assessment Scale level reached at the end of hospitalization. Patients with an apparently stable clinical picture, too, showed a change in their ability to interact with the environment. This study underlines the importance of an individualized and early cognitive-behavioral intervention protocol that can reveal minimal and fluctuating responses., (© The Author(s) 2014.)

Objective: The aim of this work was to put into evidence the long-lasting modification induced by botulinum toxin injection and rehabilitative treatment on motor control., Methods: In this contribution, we report the case of a female child showing hemiplegia, due to cerebral palsy. She underwent botulinum injection, followed by physical and occupational therapy. We quantified the biomechanical, cerebral and occupational aspects of her impaired upper limb, also dynamically, with respect to her pre- and post-treatment condition., Results: Small long-lasting improvements--induced on biomechanics by botulinum injection--triggered wide cerebral modification, well reflected in improved contextual movements and motor strategy., Conclusion: These results provide evidences that small modifications in the end-effector performance often imply cerebral modifications and improvement in finalized motor strategy.

Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50's and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis.

Despite negative blood oxygenation level dependent (BOLD) responses to visual stimuli have recently gained considerable interest, the explanation for their underlying neuronal and vascular mechanisms is still controversial. In the present study, a multimodal experimental approach is presented to shed light on the negative BOLD phenomenon in the human brain. In particular, information from functional magnetic resonance imaging (fMRI) and near infrared spectroscopy (NIRS) was integrated to confirm and gain insight into the phenomenon of negative BOLD responses (NBRs) to unpatterned intermittent photic stimulation (IPS) in healthy subjects. Eight healthy subjects participated in the study. Consistent findings emerged from the activation analysis of fMRI and NIRS data and the comparison of BOLD and hemoglobin responses at the single channel level showed that NBRs are related to a decrease in oxyhemoglobin (HbO) combined with a lower increase in deoxyhemoglobin (HHb), corresponding to a decrease in total hemoglobin (THb) and estimated cerebral blood volume (CBV). The HbO and HHb variations were significant in at least one channel in six subjects out of eight (p<0.05). The NIRS technique allowed obtaining valuable information on the vascular determinants of the NBRs, since the discrimination between HbO, HHb and THb information provided a more comprehensive view of the negative BOLD phenomenon. The within and between subject heterogeneous BOLD-Hb temporal relations pave the way to further investigations into the neurovascular properties of NBRs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the combined effect of robotic-aided gait training (RAGT) and physical therapy (PT) on functional abilities and gait pattern in children and adolescents exiting acquired brain injury (ABI), through functional clinical scales and 3D-Gait Analysis (GA)., Methods: A group of 23 patients with ABI underwent 20 sessions of RAGT in addition to traditional manual PT. All the patients were evaluated before and after the training by using the Gross Motor Function Measures (GMFM) and the Functional Assessment Questionnaire. Ambulant children were also evaluated through the 6 Minutes Walk Test (6MinWT) and GA. Finally, results were compared with those obtained from a control group of ABI children who underwent PT only., Results: After the training, the GMFM showed significant improvement in both dimensions 'D' (standing) and 'E' (walking). In ambulant patients the 6MinWT showed significant improvement after training and GA highlighted a significant increase in cadence, velocity and stride length. Moreover, hip kinematics on the sagittal plane revealed a statistically significant increase in range of motion (ROM) during the whole gait cycle, increased hip extension during terminal stance and increased ROM during the swing phase., Conclusions: The data suggest that the combined programme RAGT + PT induces improvements in functional activities and gait pattern in children and adolescents with ABI and demonstrated it to be an elective tool for the maintenance of the patients' full compliance throughout the rehabilitative programme.

We assessed abnormalities of brain functional magnetic resonance imaging (fMRI) activity during a sustained attention task (Conners' Continuous Performance Test (CCPT)) in 20 right-handed pediatric acquired brain injury (ABI) patients versus 7 right-handed age-matched healthy controls, and we estimated the correlation of such abnormalities with clinical and cognitive deficits. Patients underwent the Wechsler Intelligence Scale for Children (WISC), Wisconsin Card Sorting Test, and Functional Independence Measure (FIM) evaluations. During fMRI, patients and controls activated regions of the attention network. Compared to controls, ABI patients experienced a decreased average fMRI recruitment of the left cerebellum and a decreased deactivation of the left anterior cingulate cortex. With increasing task demand, compared to controls, ABI patients had an impaired ability to increase the recruitment of several posterior regions of the attention network. They also experienced a greater activation of frontal regions, which was correlated with worse performance on FIM, WISC, and fMRI CCPT. Such abnormal brain recruitment was significantly influenced by the type of lesion (focal versus diffuse axonal injury) and time elapsed from the event. Pediatric ABI patients experienced an inability to optimize attention network recruitment, especially when task difficulty was increased, which likely contributes to their clinical and cognitive deficits.

Upper motoreuron lesions (UML) affects people of all ages and conditions and is a major cause of disability in the young. Whereas active exercise is recognised as paramount to restore the lost motor functions, passive mobilisation of the affected limbs is regarded as a means to safeguard muscular tissue properties during a period of disuse and lack of voluntary control, which often characterises the acute and sub-acute phases. The purpose of the present work is to study the cortical reactivity in UML patients who are treated for two weeks with a robotic passive ankle mobiliser, and the clinical effects of this treatment. The rationale is that, if passive mobilisation can affect positively the functional reorganisation at a cortical level, it could be proposed as a suitable tool to maintain afferentation and guide central nervous remapping, thus bridging the period of time when active exercise is impossible due to acute paresis. Preliminary results on 7 patients (aged 15.35±4.36) showed that this therapy is very well tolerated and suggest that its application could specifically improve ankle PROM and plantarflexor muscle length. EEG data showed improved desynchronisation in at least one frequency band in 3 patients of the study, thus confirming the effects of passive mobilisation on the cortical re-organisation of some patients having UML.

Particulate matter (PM) exposure is related to pulmonary and cardiovascular diseases, with increased inflammatory status. The release of the proinflammatory interleukin- (IL-) 1β, is controlled by a dual pathway, the formation of inactive pro-IL-1β, through Toll-like receptors (TLRs) activation, and its cleavage by NLRP3 inflammasome. THP-1-derived macrophages were exposed for 6 h to 2.5  μg/cm(2) of Milan PM10, and the potential to promote IL-1β release by binding TLRs and activating NLRP3 has been examined. Summer PM10, induced a marked IL-1β response in the absence of LPS priming (50-fold increase compared to unexposed cells), which was reduced by caspase-1 inhibition (91% of inhibition respect summer PM10-treated cells) and by TLR-2 and TLR-4 inhibitors (66% and 53% of inhibition, resp.). Furthermore, summer PM10 increased the number of early endosomes, and oxidative stress inhibition nearly abolished PM10-induced IL-1β response (90% of inhibition). These findings suggest that summer PM10 contains constituents both related to the activation of membrane TLRs and activation of the inflammasome NLPR3 and that TLRs activation is of pivotal importance for the magnitude of the response. ROS formation seems important for PM10-induced IL-1β response, but further investigations are needed to elucidate the molecular pathway by which this effect is mediated.

The hemodynamic response to motor activation was investigated by time-resolved NIRS in healthy subjects and patients with unilateral impairment in motor ability. Healthy subjects performed a simple and a complex finger movement task, patients a handgrip task. A General Linear Model approach (GLM) was applied during NIRS data processing. In general, compared to the integral (continuous wave signal), higher significance of activation was found for the variance signal that selectively represents changes in the deep compartment. A discussion of GLM results with respect to task complexity and difficulty is provided.

Functional Magnetic Resonance Imaging (fMRI) in combination with Near Infrared Spectroscopy (NIRS) is finding widespread use in the analysis of brain function. While most of the studies deal with the detection of positive responses, here we focus on negative responses to visual stimulation. In a group fMRI study on Intermittent Photic Stimulation (IPS) we detected a sustained Negative BOLD Response (NBR) in the extrastriate visual cortex. To confirm and better characterize NBR, we repeated the same protocol during NIRS recordings. In this paper we show fMRI results and demonstrate the NBR on the basis of NIRS findings.

Atherosclerosis is an inflammatory disease characterized by immunological activity, in which endothelial dysfunction represents an early event leading to subsequent inflammatory vascular damage. We investigated gene expression of the adhesion molecules (AMs) ICAM-1, VCAM-1, and β1-integrin in endothelial cells (ECs) isolated from venous blood (circulating EC, cEC) and purified from femoral plaques (pEC) obtained from 9 patients with peripheral artery disease (PAD) submitted to femoral artery thrombendarterectomy (FEA). In addition, in peripheral blood mononuclear cells (PBMCs) of the same subjects, we investigated gene expression of IFN-γ, IL-4, TGF-β, and IL-10. Patients were longitudinally evaluated 1 month before surgery, when statin treatment was established, at the time of surgery, and after 2 and 5 months. All AM mRNA levels, measured by means of real-time PCR, in cEC diminished during the study, up to 41-50% of initial levels at followup. AM mRNA expression was significantly higher in pEC than in cEC. During the study, in PBMCs, TGF-β and IL-10 mRNA levels remained unchanged while IFN-γ and IL-4 levels increased; however, the ratio IFN-γ/IL-4 showed no significant modification. In PAD patients, FEA and statin treatment induce a profound reduction of AM expression in cEC and affect cytokine mRNA expression in PBMCs.

Lower limb rehabilitation is a fundamental part of post-acute care in neurological disease. Early commencement of active workout is often prevented by paresis, thus physical treatment may be delayed until patients regain some voluntary command of their muscles. Passive mobilization of the affected joints is mostly delivered in order to safeguard tissue properties and shun circulatory problems. The present paper investigates the potential role of early passive motion in stimulating cortical areas of the brain devoted to the control of the lower limb. An electro-mechanical mobilizer for the ankle joint (Toe-Up!) was implemented utilizing specially-designed shape-memory-alloy-based actuators. This device was constructed to be usable by bedridden subjects. Besides, the slowness and gentleness of the imparted motion, make it suitable for patients in a very early stage of their recovery. The mobilizer underwent technical checks to confirm reliability and passed the required safety tests for electric biomedical devices. Four healthy volunteers took part in the pre-clinical phase of the study. The protocol consisted in measuring of brain activity by EEG and NIRS in four different conditions: rest, active dorsiflexion of the ankle, passive mobilization of the ankle, and assisted motion of the same joint. The acquired data were processed to obtain maps of cortical activation, which were then compared. The measurements collected so far show that there is a similar pattern of activity between active and passive/assisted particularly in the contralateral premotor areas. This result, albeit based on very few observations, might suggest that passive motion provides somatosensory afferences that are processed in a similar manner as for voluntary control. Should this evidence be confirmed by further trials on healthy individuals and neurological patients, it could form a basis for a clinical use of early passive exercise in supporting central functional recovery.

Near Infrared Spectroscopy (NIRS) was employed for the detection of possible residual functional activations in two patients in minimally conscious state. An "ad hoc" protocol for somatosensory and motor stimulations was created and administered to the patients, synchronously to NIRS recordings. One healthy subject was also assessed with the same task for comparison. Results from the healthy subject globally agree with the literature. Moreover, we could obtain significant results from the patients data. Indeed, in one patient, the NIRS channels showing activation completely correspond to regions of residual cortex underneath. In the second patient, though, together with possible residual intact cortex insulae, some channels match large cystic formations, with fluid gathering.

Impaired hand function is often the most disabling symptom in children with hemiplegic cerebral palsy (CP). Literature provides a wide number of studies dealing either with the kinematics or the cerebral correlates of the impairment. Nevertheless, few studies exist merging both aspects together. The aim of this study is the integrated analyses of time and spatial parameters of both the affected and less-affected sides and of the EEG signal, recorded during the movement execution, for the quantitative description of the pointing gesture in children with CP. The participants (pathological and control subjects) were asked to execute a pointing task simultaneously with the recording by an optoelectronic system and an electroencephalographer. Kinematic data were processed for the extraction of several synthetic indexes, to be correlated with parameters derived from frequency analysis of the electroencephalographic signal. Kinematic results showed statistical differences (1) between the affected and the less-affected arms in patients and (2) between the less-affected arm in patients and the normal arm in controls. Further differences were found in kinematics with respect to bilateral or ipsilateral motor control, extracted from EEG. Given the different behavior evidenced by either ipsilateral or contralateral reorganization, and considering the role of feedback and feed-forward contributions to motor programming, some hypotheses emerged about the motor control during pointing task in CP.

The benzothiazinone BTZ043 is a tuberculosis drug candidate with nanomolar whole-cell activity. BTZ043 targets the DprE1 catalytic component of the essential enzyme decaprenylphosphoryl-β-D-ribofuranose-2'-epimerase, thus blocking biosynthesis of arabinans, vital components of mycobacterial cell walls. Crystal structures of DprE1, in its native form and in a complex with BTZ043, reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighboring catalytic lysine residue. Kinetic studies confirm that BTZ043 is a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labeled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the flavin adenine dinucleotide cofactor in DprE1 and may be the natural electron acceptor for this reaction in the mycobacterium. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors.

Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette-Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-β-D: -ribose 2'-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of D: -arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen's survival.

We propose an experimental protocol for the integrated study of motor adaptation during target-based movements. We investigated how motor adaptation affects both cerebral activity and motor performance during the preparation and execution of a pointing task, under different conditions of external perturbation. Electroencephalography (EEG) and movement analysis were simultaneously recorded from 16 healthy subjects enrolled in the study. EEG signal was preprocessed by means of independent component analysis and empirical mode decomposition based Hilbert Huang transform, in order to extract event-related synchronization (ERS) and desynchronization (ERD) parameters. Movement analysis provided several kinematic indexes, such as movement durations, average jerk, and inter-quartile-ranges. Significant correlations between score, neural, and kinematic parameters were found. Specifically, the duration of the going phase of movement was found to correlate with synchronization in the beta brain rhythm, in both the planning and executive phases of movement. Inter-quartile ranges and average jerk showed correlations with executive brain parameters and ERS/ERDcueBeta, respectively. Results indicate the presence of links between the primary motor cortex and the farthest ending point of the upper limb. In the present study, we assessed significant relationship between neural and kinematic descriptors of motor adaptation, during a protocol requiring short-term learning, through the modulation of the external perturbations.

We evaluated frontal brain activation during a mixed attentional/working memory task with graded levels of difficulty in a group of 19 healthy subjects, by means of time-domain functional near-infrared spectroscopy (fNIRS). Brain activation was assessed, and load-related oxy- and deoxy-hemoglobin changes were studied. Generalized linear model (GLM) was applied to the data to explore the metabolic processes occurring during the mental effort and, possibly, their involvement in short-term memorization. GLM was applied to the data twice: for modeling the task as a whole and for specifically investigating brain activation at each cognitive load. This twofold employment of GLM allowed (1) the extraction and isolation of different information from the same signals, obtained through the modeling of different cognitive categories (sustained attention and working memory), and (2) the evaluation of model fitness, by inspection and comparison of residuals (i.e., unmodeled part of the signal) obtained in the two different cases. Results attest to the presence of a persistent attentional-related metabolic activity, superimposed to a task-related mnemonic contribution. Some hemispherical differences have also been highlighted frontally: deoxy-hemoglobin changes manifested a strong right lateralization, whereas modifications in oxy- and total hemoglobin showed a medial localization. The present work successfully explored the capability of fNIRS to detect the two neurophysiological categories under investigation and distinguish their activation patterns.

The potential disturbance in the prefrontal cortex hemodynamic signal measured by functional near infrared spectroscopy (NIRS), due to forehead skin flowmotion, detected by laser Doppler flowmetry, was investigated by a standard protocol of hemodynamic challenge by Valsalva maneuver, aimed at assessing and disentangling local regulatory responses in skin vasomotion and in cerebral perfusion in presence of a strong systemic drive, and to quantify the common information in the two signals. The deep cortical NIRS signal did not appear to be affected by surface vasomotor activity, and autoregulation dynamics were dominant with respect to autonomic control of circulation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-β-D-ribofuranose 2'-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a near-quantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents., (© 2011 American Chemical Society)

Aging is associated to a decline in immune functions that are in part related to a defective protein kinase C dependent signal transduction machinery. RACK-1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. We have previously demonstrated, in the elderly, a defective PCKβII (Protein Kinase C βII) translocation related to a decrease in RACK-1 protein expression, which is correlated to the age-associated decline in DHEA (dehydroepiandrosterone) levels. As a consequence of this signal transduction impairment, a significant decrease in immune cells functionality was observed. Furthermore, we could demonstrate that in vivo and in vitro DHEA administration restored RACK-1 level and immune functions, indicating that this hormone behaved as a positive RACK-1 regulator. We have most recently characterized the human GNB2L1 promoter region, coding for RACK-1 protein. Although no direct DHEA responsive elements were found, a glucocorticoid responsive element (GRE) was identified. The purpose of this work was to investigate, in the human pro-myelocytic cell line THP-1, whether physiological cortisol concentrations were able to modulate GNB2L1 promoter activity, RACK-1 transcription as well as cytokine production. As DHEA is endowed of anti-glucocorticoid properties in several cellular systems, and as cortisol:DHEA ratio imbalance is relevant in aging, we also investigated their possible interaction at the RACK-1 expression level. We could demonstrate that cortisol acted in a dose-related manner as a GNB2L1 promoter repressor, reducing RACK-1 mRNA expression and protein level. Probably by interfering with glucocorticoid receptor binding to GRE sequence, prolonged DHEA exposure counteracted cortisol effects, restoring RACK-1 levels and cytokine production, as assessed by LPS-induced TNF-α release., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We evaluated neurovascular and autonomic response to a Divided Attention task within a group of 16 healthy subjects, by means of Electroencephalography, Electrocardiography, functional Near Infrared Spectroscopy techniques, acquired simultaneously. We exctracted Alpha (8-13,5 Hz) and Beta (13,5-30 Hz) power rhythms with a spectral autoregressive residual model, and inter-beat-interval (RR series) and separated superficial (extracortical) and depth NIRS contribution. Cross Correlation Function at different time lags was then calculated between each signal and the task, modeled as a square wave and among couples of signals, in order to evaluate the sequence of activation of the different physiological districts involved and the common information shared. Results showed the presence of a cascade of responses and a strong influence by the block task on each signal, representative of the neurovascular coupling elicited by the cognitive cerebral activation.

The aminoglycosidic antibiotic hygromycin B presents a peculiar chemical structure, characterized by two sugar rings joined via a spiro connection. The Cu(ii) complex of hygromycin B in water solution was characterized by (1)H-NMR, UV-Vis, EPR and CD spectroscopy, combined with potentiometric measurements. The spin-lattice relaxation enhancements were interpreted by the Solomon-Bloembergen-Morgan theory, allowing us to calculate copper-proton distances that were used to build a model of the complex by molecular mechanics and dynamics calculations. The fidelity of the proposed molecular model was checked by ROESY maps. Moreover DNA damage by the Cu(ii)-hygromycin B system was also investigated, showing single and double strand scissions exerted by the complex at concentrations in the range 1-5 mM. Addition of either hydrogen peroxide or ascorbic acid to each sample resulted in the shift of the cleavage potency towards lower concentrations of the complex.

The aim of this work was to design a multimodal approach for the investigation of the different factors Motor Learning involves (e.g. planning, execution, correction, …). Attention was focused onto the issue of Motor Adaptation (MA), which takes place when learning of a partially (but not completely) new motor strategy is needed. We hereby present a report about the methodological set-up which has been developed and tested for the investigation of motor adaptation during repetitive pointing tasks. We exploited the potentialities of the combined use of electroencephalographic and motion analysis techniques to find possible relation between the activity of the central nervous system and the neuro-musculo-skeletal one. The adaptation-related changes in oscillatory brain activity and movement kinematics were monitored during a visually-guided, feedback-controlled, sequence of pointing tasks. Our preliminary results suggest that the proposed protocol: (i) can discern modifications of brain activity in the alpha and beta frequency bands; (ii) is sensible to kinematic alterations; and, (iii), allows a quantitative evaluation of performance in terms of both final result and motor patterns. In this work we identified a bunch of possible neuro-motor biomarkers, which we propose as possible indicators of adaptation.

Physiological neural mechanisms underlying attentional processes were investigated by means of moving dipoles method in survivors of diffuse axonal injury (DAI) and in a control group. N2 and P3 components were obtained by means of the administration of the Conner's Continuous Performance Test. Analysis of behavioral responses showed significant between-group differences in reaction times and error rates. A different number of dipoles was needed for the reconstruction of both N2 and P3 components in the two groups. The study of dipoles location revealed major displacements in damaged brains with respect to physiology. In conclusion, data from DAI patients showed that neuronal reorganization following brain injury evolves towards the generation of multiple central sources.

Purpose: Sustained attention is one of the most important cognitive abilities for the management of everyday life, but it is often studied only at the behavioral level, while functional correlates are scarcely investigated. In this article, the authors address the topic of characterizing the dynamics of cerebral metabolism in the prefrontal cortex during a task of prolonged attention., Method: By means of multichannel time-resolved functional near-infrared spectroscopy and generalized linear model based data processing, the authors measured the hemodynamic response of the prefrontal cortex from 19 healthy subjects to a shortened version of a sustained attention task (Conners' Continuous Performance Test), lasting for 10 min., Results: The task elicited significant brain activation, which did not remain constant for the entire task, but showed a drop not correlated with performance decay 4 min after the beginning of the task. Furthermore, oxygenated hemoglobin showed an increasing trend also during the first phase of the recovery, just after the end of the task., Conclusion: The results indicate a nontrivial dynamics of neural activation, habituation processes, and hemodynamic/metabolic coupling. These results encourage further studies about continuous stimulation of cognitive functions on both healthy and pathological subjects.

The homeostasis of metal ions, especially copper and zinc, is a major factor that may influence the prion diseases and the biological function of prion protein (PrP). The His-rich regions are basic sites for metal binding and antioxidant activity of the PrP structures. Animal prion-like proteins contain also His-rich domains, and their coordination chemistry may provide better insight into the chemistry and biology of PrP structures and related diseases. Herein, we report an equilibrium study on heteronuclear Zn(2+)-Cu(2+) complexes with zrel-PrP fragments from zebrafish. Potentiometric, spectroscopic, and mass spectrometric methods showed that the binding of copper is much more effective than the binding of zinc. At physiological pH, both metals bind to the histidine imidazole N donors of the studied peptides.

The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).

In clinical routine, the evaluation of sustained attention is often performed by analyzing the behavioral data collected during specific tests. Such analyses are rarely accompanied by a detailed examination of the subject's simultaneous electroencephalographic (EEG) activity, and particularly its frequency content. In this study, a group of healthy volunteers and a group of patients affected by diffuse axonal injury (DAI) were tested while performing a modified version of the Conners' continuous performance test. A comparative study was carried out between the behavioral and neuropsychological data obtained during the task, to investigate neural activation. Spectral power was calculated for each of the recorded EEG signals, taking account of the frequency bands traditionally considered in literature. Then a compressed spectral array sequence of spectra was plotted to put into evidence the temporal modifications in the signal power spectral density, and, finally, the analysis of the rhythm variability was carried out. Evaluation of the results thus obtained shows that the two groups registered very different cerebral activation dynamics during the ongoing attentional task. Moreover, DAI patients showed mild cortical activation in the prefrontal region, spread equally throughout both brain hemispheres, while controls showed strong predominant activation of the right prefrontal area. Our findings encourage further investigations of the combined employment of tests and EEG recordings during the clinical assessment of sustained attention performance.

Cyclosporin A (CsA) is a cyclic naturally occurring peptide used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is due to the formation of a complex with cyclophilin A (Cyp), in which the cis 9MeLeu-10MeLeu amide bond of CsA assumes a trans conformation. The mechanism of the conformational inversion has not been delineated, but it has been postulated that metal ions binding induces a conformational change that enables CsA to bind Cyp. In this work, we solved the structures of CsA in sodium dodecyl sulfate (SDS) micelles (which enhance its solubility and mimic the hydrophobic environment clinically used for drug delivery) and its complex with Dy(III) ion, whose coordination chemistry is frequently used to reproduce the effect of Ca(II). The paramagnetic properties of Dy(III) allowed us to build up a structure using proton relaxation enhancements, which remains stable in a MD simulation in the micelle environment.

The interaction between Cu(II) and the rat amyloid beta (1-28) fragment in micellar solutions at pH 7.5 was investigated by CD and NMR spectroscopy; the proton-copper distances were used in restrained molecular dynamics simulations to obtain a structural model of the Cu(II) complex.

Alzheimer's disease is a fatal neurodegenerative disorder involving the abnormal accumulation and deposition of peptides (amyloid-beta, Abeta) derived from the amyloid precursor protein. Here, we present the structure and the Zn2+ binding sites of human and rat Abeta(1-28) fragments in water/sodium dodecyl sulfate (SDS) micelles by using 1H NMR spectroscopy. The chemical shift variations measured after Zn2+ addition at T>310 K allowed us to assign the binding donor atoms in both rat and human zinc complexes. The Asp-1 amine, His-6 Ndelta, Glu-11 COO-, and His-13 Nepsilon of rat Abeta28 all enter the metal coordination sphere, while His-6 Ndelta, His-13, His-14 Nepsilon, Asp-1 amine, and/or Glu-11 COO- are all bound to Zn2+ in the case of human Abeta28. Finally, a comparison between the rat and human binding abilities was discussed.

The solution structure of kanamycin A interacting with a ribosomal A-site fragment was solved by transferred-NOE techniques and found to agree with the structure of the complex observed in the crystal. Despite the fast exchange conditions found for the interaction, the bound form was identified by NOESY spectroscopy. At 600 MHz, NOE effects are only observed for the RNA-associated antibiotic. Dissociation constants were measured by NMR spectroscopy for two sites of interaction (K(d1)=150+/-40 microM; K(d2)=360+/-50 microM). Furthermore, the effects of the Cu(II) ion on the antibiotic, on the RNA fragment that mimics the bacterial ribosomal A site, and on the complex formed between these two entities were analyzed. The study led to the proposal of a model that localizes the copper ion within the kanamycin-RNA complex.

We evaluated frontal brain activation during a Working Memory task with graded levels of difficulty in a group of 15 healthy subjects, by means of fNIRS technique. Brain activation (% signal change with respect to the baseline) was computed and load-related increases of blood oxygenation were found for the four different levels of task difficulty. Generalized Linear Models were applied to the data in order to evaluate the metabolic processes occurring during the mental effort and, possibly, their involvement in short term memorization. The Cognitive Failure Questionnaire was also administered in order to assess its diagnostic relevance in clinics. Results attest a monotous increase of brain oxygenation with WM load during the first half of task, persisting through the central resting period and prevailing in the left hemisphere. Decreasing WM load is also coupled with oxygenated hemoglobin decrease.

Electrophysiological studies provide useful information for diagnosis and classification of myoclonus, and for the investigation of its generative mechanisms, due to association of myoclonus with abnormally increased excitability of cortical structures. In this work we analyzed the polygraphic data of a 7-year old girl affected by continuous partial epilepsy with focal myoclonus both related and not related with epileptiform discharges on EEG. We applied Sample Entropy (SampEn) and Lempel-Ziv complexity (LZ) methods to investigate the regularity and complexity content of EEG recordings and to find possible analogies in the behaviour of non-parametric complexity measures in epilepsy and in myoclonus. Our results show that these algorithms succeeded in finding a significant difference between the hypothesized focus on C3 electrode and the contralateral electrode C4, for EEG correlated myoclonus. A significant difference between the two contralateral electrodes (C3-C4) was also found for non EEG correlated myoclonus, but only by means of SampEn. This preliminary study confirmed the ability of entropic methods in discriminating myoclonic events. Indeed, near the myoclonic focus location both SampEn and LZ methods showed below average values.